Factor V Leiden mutation does not affect coagulopathy or outcome in lethal H1N1 influenza.

Influenza A is a major cause of mortality. Knowledge on coagulation activation in influenza infection is limited. The factor V Leiden (FVL) mutation is possibly subject to positive selection pressure. It is unknown whether this mutation impacts on the outcome of severe influenza. In the present study, the effect of lethal influenza on pulmonary and systemic coagulation activation and whether or not FVL mutation alters coagulation activation in and the course of lethal influenza, was determined. Wild-type mice, and mice heterozygous or homozygous for FVL were infected intranasally with a lethal dose of H1N1 (haemagglutinin 1 and neuraminidase 1) influenza A. Mice were sacrificed after 48 or 96 h for determination of coagulation activation, histopathology, pulmonary inflammatory parameters and viral load, or were observed in a survival study. Extensive local and systemic coagulation activation during lethal influenza was demonstrated by increased lung and plasma levels of thrombin-antithrombin complexes and fibrin degradation products, and by pulmonary fibrin deposition. FVL mutation did not influence the procoagulant response, lung histopathology or survival. FVL mice demonstrated elevated viral loads 48 h after infection. In conclusion, coagulation is activated locally and systemically during lethal murine influenza A infection. The FVL mutation does not influence coagulation activation, lung inflammation or survival in lethal influenza A.

Activation of coagulation with concurrent impairment of anticoagulant mechanisms correlates with a poor outcome in severe melioidosis.

BACKGROUND: Melioidosis, which is caused by infection with the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of sepsis in South-East Asia with a mortality of up to 40%. Knowledge of the involvement of coagulation and fibrinolysis in the pathogenesis of melioidosis is highly limited. OBJECTIVE: To define the involvement of the coagulation and fibrinolytic systems in patients with severe melioidosis. METHODS: Parameters of coagulation and fibrinolysis were measured in 34 patients with culture proven septic melioidosis and 32 healthy controls. RESULTS: Patients demonstrated strong activation of the coagulation system, as reflected by high plasma levels of soluble tissue factor, the prothrombin fragment F(1+2) and thrombin-antithrombin complexes (TATc), and consumption of coagulation factors resulting in a prolonged prothrombin time and activated partial thromboplastin time. Concurrently, anticoagulant pathways were downregulated in patients: protein C, protein S, and antithrombin levels were all decreased when compared to controls. Patients also demonstrated evidence of activation and inhibition of fibrinolysis, as reflected by elevated concentrations of tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1, plasmin-alpha2-antiplasmin complexes (PAPc) and D-dimer. High TATc/PAPc ratios in patients pointed to a predominance of the prothrombotic pathway in melioidosis. Furthermore, soluble thrombomodulin levels were increased. The extent of coagulation activation correlated with mortality; patients who went on to die had higher TATc, F(1+2), tPA and PAPc and lower protein C and antithrombin levels on admission than patients who survived. CONCLUSIONS: The coagulation system is strongly activated during melioidosis. A high degree of activation of the coagulation system is an indicator of poor outcome in patients with melioidosis.

LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction.

LPS-binding protein (LBP) and serum lipoproteins cooperate in reducing the toxic properties of LPS. In the present study, we demonstrate that LBP circulates in association with LDL and VLDL in healthy persons. ApoB was found to account at least in part for the interaction of LBP with LDL and VLDL. Although LBP interacted with purified apoA-I in vitro, no association of LBP with apoA-I or HDL was found in serum. Consistent with the observed association of LBP with LDL and VLDL, these lipoproteins also were demonstrated to be the predominant LPS-binding lipoproteins. Most interestingly, the association of LBP with LDL and VLDL strongly enhanced the capacity of these lipoproteins to bind LPS. Because this function of LBP is of utmost importance during infection, the association of LBP and LPS with lipoproteins was also studied in serum from septic patients. In septic serum containing high LBP levels and a markedly altered lipoprotein spectrum, most of the LBP is associated with LDL and VLDL, although some LBP appeared to circulate free from lipoproteins. Also in this serum, LPS was found to bind predominantly to LDL and VLDL. The observed binding of LBP and LPS to LDL and VLDL, as well as the LBP-dependent incorporation of LPS into these lipoproteins, emphasizes a crucial role for circulating LBP-LDL/VLDL complexes in the scavenging of LPS.

Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Identification of Salp15 homologues in Ixodes ricinus ticks.

The 15-kDa Ixodes scapularis salivary gland protein Salp15 protects Borrelia burgdorferi sensu stricto from antibody-mediated killing and facilitates infection of the mammalian host. In addition, Salp 15 has been shown to inhibit T-cell activation. We determined whether Ixodes ricinus, the major vector for Lyme borreliosis in Western Europe, also express salp15-related genes. We show that engorged I. ricinus express salp15 and we have identified three Salp15 homologues within these ticks by reverse transcriptase-polymerase chain reaction (RT-PCR). One of the predicted proteins showed 80% similarity to I. scapularis Salp15, evenly distributed over the entire amino acid sequence, whereas the two other predicted proteins showed approximately 60% similarity, mainly confined to the signal sequence and C-terminus. Comparison of the DNA and protein sequences with those deposited in several databases indicates that these proteins are part of a Salp15 family of which members are conserved among different Ixodes species, all capable of transmitting B. burgdorferi sensu lato. This suggests that these Salp15 homologues could also play a role in the transmission of diverse Borrelia species and in inhibition of T-cell activation.

Thrombin contributes to protective immunity in pneumonia-derived sepsis via fibrin polymerization and platelet-neutrophil interactions.

Essentials Immunity and coagulation are linked during sepsis but the role of thrombin is not fully elucidated. We investigated the effect of thrombin inhibition on murine Klebsiella pneumosepsis outcome. Thrombin is crucial for survival and limiting bacterial growth in pneumonia derived sepsis. Thrombin improves host defense via fibrin and enhancement of platelet-neutrophil interactions. SUMMARY: Background Innate immunity and coagulation are closely linked during sepsis. Their interaction can be detrimental to the outcome because of microvascular failure but can also enhance host defense. The role of thrombin therein has not been fully elucidated. Objective We aimed to investigate the contribution of thrombin to the host response during pneumonia-derived sepsis. Methods Mice treated with the specific thrombin inhibitor dabigatran or control chow were infected with the common human sepsis pathogen Klebsiella (K.) pneumoniae via the airways. In subsequent infection experiments, mice were additionally treated with ancrod to deplete fibrinogen. Ex vivo Klebsiella growth was assessed by incubating human whole blood or specific blood components in various conditions with Klebsiella. Results Thrombin inhibition by dabigatran enhanced bacterial outgrowth and spreading, and accelerated mortality. Thrombin inhibition did not influence neutrophil recruitment to the lung or activation or neutrophil extracellular trap formation. Dabigatran reduced D-dimer formation and fibrin deposition in the lung. Fibrin depletion also enhanced bacterial outgrowth and spreading, and thrombin inhibition had no additional effect. Both thrombin and fibrin polymerization inhibited ex vivo Klebsiella outgrowth in human whole blood, which was neutrophil dependent, and the effect of thrombin required the presence of platelets and platelet protease activated receptor-1. In vivo thrombin inhibition reduced platelet-neutrophil complex formation and endothelial cell activation, but did not prevent sepsis-induced thrombocytopenia or organ damage. Conclusions These results suggest that thrombin plays an important role in protective immunity during pneumonia-derived sepsis by fibrin polymerization and enhancement of platelet-neutrophil interactions.

Functional protection by acute phase proteins alpha(1)-acid glycoprotein and alpha(1)-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation.

BACKGROUND: Ischemia followed by reperfusion (I/R) causes apoptosis, inflammation, and tissue damage leading to organ malfunction. Ischemic preconditioning can protect against such injury. This study investigates the contribution of the acute phase proteins alpha(1)-acid glycoprotein (AGP) and alpha(1)-antitrypsin (AAT) to the protective effect of ischemic preconditioning in the kidney. METHODS AND RESULTS: Exogenous AGP and AAT inhibited apoptosis and inflammation after 45 minutes of renal I/R in a murine model. AGP and AAT administered at reperfusion prevented apoptosis at 2 hours and 24 hours, as evaluated by the presence of internucleosomal DNA cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and the determination of renal caspase-1- and caspase-3-like activity. AGP and AAT exerted anti-inflammatory effects, as reflected by reduced renal tumor necrosis factor-alpha expression and neutrophil influx after 24 hours. In general, these agents improved renal function. Similar effects were observed when AGP and AAT were administered 2 hours after reperfusion but to a lesser extent and without functional improvement. Moreover, I/R elicited an acute phase response, as reflected by elevated serum AGP and serum amyloid P (SAP) levels after 24 hours, and increased hepatic acute phase protein mRNA levels after 18 hours of renal reperfusion. CONCLUSIONS: We propose that the antiapoptotic and anti-inflammatory effects of AGP and AAT contribute to the delayed type of protection associated with ischemic preconditioning and other insults. This mechanism is potentially involved in the course of many clinical conditions associated with I/R injury. Moreover, exogenous administration of these proteins may provide new therapeutic means of treatment.

Platelet and endothelial cell P-selectin are required for host defense against Klebsiella pneumoniae-induced pneumosepsis.

BACKGROUND: Sepsis is associated with activation of platelets and endothelial cells accompanied by enhanced P-selectin surface expression. Both platelet- and endothelial P-selectin have been associated with leukocyte recruitment and induction of inflammatory alterations. Klebsiella (K.) pneumoniae is a common human sepsis pathogen, particularly in the context of pneumonia. METHODS: Wild-type (WT) and P-selectin-deficient (Selp(-/-) ) mice or bone marrow chimeric mice were infected with K. pneumoniae via the airways to induce pneumosepsis. Mice were sacrificed during early (12 h after infection) or late-stage (44 h) sepsis for analyses, or followed in a survival study. RESULTS: Selp(-/-) mice displayed 10-1000-fold higher bacterial burdens in the lungs, blood and distant organs during late-stage sepsis. P-selectin deficiency did not influence leukocyte recruitment to the lungs, but was associated with decreased platelet-monocyte complexes and increased cytokine release. Bone marrow transfer studies revealed a role for both platelet and endothelial cell P-selectin as mice deficient in platelet or endothelial cell P-selectin displayed an intermediate phenotype in bacterial loads and survival compared with full wild-type or full knockout control mice. CONCLUSION: Both platelet and endothelial cell P-selectin contribute to host defense during Klebsiella pneumosepsis.

The role of platelet MyD88 in host response during gram-negative sepsis.

BACKGROUND: Beside their role in hemostasis, platelets serve as sentinel cells in host defense during infection. In sepsis, platelets have been implicated in both beneficial (antibacterial) and detrimental responses (thrombosis and organ damage). Toll-like receptors and their common adaptor, myeloid differentiation factor 88 (MyD88), are essential for pathogen recognition and protective immunity. Platelets express functional Toll-like receptors and MyD88, which participate in platelet responsiveness to bacterial agonists. OBJECTIVE: Considering the pivotal involvement of platelets and MyD88 in the host response to bacteria, we studied the role of platelet MyD88 in gram-negative sepsis using intravenous and airway infections with the common human sepsis pathogen Klebsiella pneumoniae. METHODS: Platelet-specific Myd88(-/-) mice were generated by crossing mice with a conditional Myd88 flox allele with mice expressing Cre recombinase controlled by the platelet factor 4 promoter. In a reverse approach, full Myd88(-/-) mice were transfused with wild-type platelets. RESULTS: In both settings, platelet MyD88 did not impact on bacterial growth or dissemination. In addition, platelet MyD88 did not influence hallmark sepsis responses such as thrombocytopenia, coagulation or endothelial activation, or distant organ injury. Platelet MyD88 played no role in lung pathology during pneumonia-derived sepsis. CONCLUSION: Despite known literature, platelet MyD88-dependent TLR signaling does not contribute to the host response during gram-negative sepsis.

Regulation and regulatory role of gamma-carboxyglutamic acid containing clotting factors.

Blood coagulation is initiated following damage of the vessel wall. The large number of proteins that participate in the reactions that regulate blood coagulation must behave in a concerted manner to generate alpha-thrombin. This most abundant enzyme product of the coagulation process acts on the soluble blood protein fibrinogen to create the insoluble fibrin clot, trigger platelet activation, and initiate a variety of vascular processes ranging from coagulation inhibition and activation of fibrinolysis to cell growth. The majority of enzymes that participate in the blood-clotting process circulate in blood as inactive zymogens and procofactors. Following mechanical injury to the vessel wall, a cell-associated protein, tissue factor, is exposed and initiates a series of enzymatic reactions localized on a membrane surface generally provided by cells and accumulating platelets. The binding of the enzymes and zymogen substrates to the exposed membrane surface and their activation are in part governed by the gamma-carboxyglutamic acid residues (gla) of each protein. Recently, it has been demonstrated that the gla residues are necessary for Ca2+ binding and subsequent exposure of the hydrophobic membrane binding regions to the cell bilayer. Thus, the gla residues play a preeminent role during the blood-clotting process. Absence of gamma-carboxylation of one or more of the critical glutamic acid residues results in an impaired coagulation/anticoagulation process which may lead to a bleeding diathesis or thrombosis.

The regulation of clotting factors.

Blood clotting involves a multitude of proteins that act in concert in response to vascular injury to produce the procoagulant enzyme alpha-thrombin, which in turn is responsible for the generation of the fibrin plug. However, while generation of the fibrin plug is required for the arrest of excessive bleeding, unregulated clotting will result in the occlusion of the blood vessels and thrombosis. Thus, the regulation of the delicate balance between the procoagulant and anticoagulant mechanisms is of extreme importance for survival. While the majority of proteins involved in blood coagulation circulate as inactive zymogens that require proteolytic activation in order to function, approximately 1% of the circulating factor VII molecules are active. Factor VIIa, possess a serine protease active site, has poor catalytic activity, and is not inhibited by the circulating stoichiometric protease inhibitors. Following injury to the vasculature and subsequent exposure of the integral membrane glycoprotein, tissue factor (TF), the circulating factor VIIa molecules can bind to the exposed TF forming the extrinsic tenase complex (TF/factor VIIa) and initiate the blood coagulation process. Formation of the TF/factor VIIa complex increases the catalytic efficiency of the enzyme by four orders of magnitude when compared with factor VIIa alone. This cell-associated enzymatic complex initiates a series of enzymatic reactions, leading to the generation of alpha-thrombin and ultimately to the formation of the fibrin plug. The procoagulant enzymatic complexes (i.e., prothrombinase, intrinsic tenase, and extrinsic tenase) are similar in structure and composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. While the activity of the extrinsic tenase complex is limited by the availability (exposure) of its cell-associated cofactor (TF) it is remarkable that the activities of both the prothrombinase complex (factor Va/factor Xa) as well as the intrinsic tenase complex (factor VIIIa/factor IXa) are limited by the presence of the two soluble, nonenzymatic cofactors, factor Va and factor VIIIa. Factor Va and factor VIIIa, which are very similar in structure and function, are required for prothrombinase and intrinsic tenase activities, respectively, because both cofactors express a dual function in their respective complexes, acting as an enzyme receptor and catalytic effector on the cell surface. The cofactors derive from inactive plasma precursors by regulatory proteolytic events that involve alpha-thrombin. In general, bleeding tendencies are usually associated with defects in the activation of one of the zymogens or the cofactors of the procoagulant complexes. However, the activity of all of the complexes is also limited by the availability of an adequate membrane surface provided by endothelial cells, platelets, and monocytes. The cell surface provides a site for the recruitment of the appropriate proteins and allows for fast and efficient clot formation. In the absence of an appropriate membrane surface, the procoagulant complexes have limited catalytic efficiency. Thus, timely exposure of the adequate membrane surface is an additional step in the regulation of alpha-thrombin formation. alpha-Thrombin participates in its own down-regulation by binding to the endothelial cell receptor thrombomodulin, initiating the protein C pathway, which in turn leads to the formation of activated protein C (APC). APC is required for efficient neutralization of factor Va cofactor activity, which results in the inactivation of the prothrombin-activating complex. This inactivation can only occur in the presence of the appropriate membrane surface. Thus, while following alpha-thrombin activation, factor VIIIa is rapidly and spontaneously inactivated by dissociation of the A2 domain from the rest of the cofactor, APC is required for down-regulation of alpha-thrombin formation by prothrombinase. (ABSTRACT

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.

The region Ser333-Arg356 of the alpha-chain of human C4b-binding protein is involved in the binding of complement C4b.

Human C4b-binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the alpha-chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the alpha-chain of C4BP that interacts with C4b, tryptic fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333-Arg356 of the alpha-chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the alpha-chain of C4BP is likely to be involved in the interaction with C4b.

Apoptosis and chemokine induction after renal ischemia-reperfusion.

BACKGROUND: One of the earliest prerequisites for the development of inflammation after ischemia-reperfusion (I/R) is local chemokine expression. We recently demonstrated that apoptosis, characterized by intracellular caspase-activation, contributes to the development of inflammation after I/R. METHODS: The contribution of apoptosis was investigated using the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F in a murine model of renal I/R. Renal expression of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC was studied using RT-PCR and immunohistology. Measuring myeloperoxidase activity and serum ureum and creatinine levels assessed neutrophil influx and kidney dysfunction. RESULTS: We demonstrate renal up-regulation of KC and MIP-2 after 1 to 16 hr of reperfusion. Treatment with the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F effectively prevented I/R-induced renal apoptosis, KC, and MIP-2 up-regulation after 2 hr of reperfusion as well as neutrophil influx and functional impairment after 24 hr of reperfusion. CONCLUSIONS: These data for the first time show that chemokine induction following I/R is dependent on caspase activation.

Activated caspase-1 is not a central mediator of inflammation in the course of ischemia-reperfusion.

BACKGROUND: Upon transplantation, donor organs subjected to prolonged ischemia suffer from reperfusion injury. Recent observations suggest that caspase activation is involved in inducing the deleterious inflammatory reaction that mediates reperfusion injury. Release of cytokines like interleukin (IL)-1 and IL-18 may occur during apoptosis through activation of caspase-1/IL-1beta-converting enzyme. We hypothesized that caspase-1 activation is a key event in apoptosis/ caspase-dependent inflammation during the development of renal reperfusion injury. METHODS: Caspase-1-/-, caspase-1+/+ as well as Swiss mice were subjected to 45 min of renal ischemia and 24 hr of reperfusion. Animals were administered agents capable of neutralizing the pro-inflammatory activation products of caspase-1 (IL-1 receptor antagonist, anti-IL-1 receptor antibody, and anti-IL-18 antibody). The extent of renal functional deterioration, inflammation, and apoptosis were compared. RESULTS: No improvement in renal function as reflected by serum ureum and creatinine were found in caspase-1-/- mice as compared to wild type controls. Caspase-1-/- mice showed slightly attenuated renal inflammation as indicated by decreased renal neutrophil influx, but failed to show changes in intrarenal tumor necrosis factor-alpha production. Moreover, caspase-1-/- mice clearly exhibited reperfusion-induced apoptosis as reflected by renal terminal deoxynucleotidyltransferase histology and internucleosomal DNA cleavage. Treatment with IL-1 receptor antagonist, anti-IL-1 receptor antibody, or anti-IL-18 antibody minimally reduced renal functional deterioration, inflammation, and apoptosis. CONCLUSIONS: These findings suggest that activated caspase-1 and its inflammatory products are involved in, but not crucial to, the induction of inflammation after renal ischemia-reperfusion. Hence, apart from caspase-1, other (combinations of) activated caspases are likely to be more prominently involved in renal reperfusion injury.

Contribution of platelet-derived factor Va to thrombin generation on immobilized collagen- and fibrinogen-adherent platelets.

Adhesion of platelets to immobilized collagen induces the expression of anionic phospholipids, e.g. phosphatidylserine (PS), in the outer leaflet of the plasma membrane of these platelets. In contrast, of the platelets that adhere to immobilized fibrinogen only a small sub-population representing 10 +/- 3% of the total population of the fibrinogen-adherent platelets has exposed PS as probed by annexin V binding. Although the presence of PS is thought to be critical for thrombin generation at the platelet surface, no information is available about the effect of this differential PS exposure on the ability of adherent platelets to support thrombin generation. Perfusion of the fibrinogen- or collagen-adherent platelets with solutions containing factor Xa and prothrombin resulted in thrombin generation that i) increased linear during the first perfusion minutes, ii) was about two-fold faster at collagen-adherent than at fibrinogen-adherent platelets and iii) was for more than 98% restricted to the surface of the adherent platelets. It appeared that the lower thrombin generating capacity of fibrinogen-adherent platelets is not due to a lower overall surface density of PS, but is caused by lower amounts of platelet-bound factor Va. Firstly, in both cases thrombin generation could be completely attenuated with antibodies against human factor Va, and secondly, in the presence of an excess of exogenous plasma-derived factor Va similar initial rates of thrombin formation were measured for collagen- and fibrinogen-adherent platelets. Our findings suggest a unique role for immobilized collagen in maintaining haemostasis.

Platelets and phospholipids in tissue factor-initiated thrombin generation.

The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 microM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 microM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 x 10(8)/ml to 0.5 x 10(8)/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient's blood was accelerated from approximately 10 min to 5 min when the platelet concentration increased from 0.05 x 10(8)/ml to 0.11 x 10(8)/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 x 10(8)/ml (range 0.5-3.1 x 10(8)/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 x 10(8)/ml to 3.1 x 10(8)/ml (4.0+/-0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 x 10(8)/ml.

The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S.

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in plasma and in purified systems. This anticoagulant effect of protein S can be explained either by a direct interaction of protein S with one of the components of the complexes and/or by the interference with the binding of these components to phospholipid surfaces. During our investigation we noted that protein S preparations purified in different ways and derived from different sources, expressed discrepant APC cofactor and direct anticoagulant activity. In order to elucidate these differences and to study the mechanism of the APC-independent activity of protein S, we compared the protein S preparations in phospholipid-binding properties and anticoagulant activity. The dissociation constant for the binding of protein S to immobilized phospholipids ranged from 7 to 74 nM for the different protein S preparations. APC-independent inhibition of both prothrombinase and tenase activity performed on phospholipid vesicles and in plasma showed a strong correlation with the affinity for phospholipids. The APC-independent activity could be abolished by monoclonal antibodies that were either calcium-dependent and/or directed against epitopes in the Gla-region of protein S, suggesting that the protein S-phospholipid interaction is crucial for the APC-independent anticoagulant function of protein S. Protein S preparations with a low APC-independent activity expressed a high APC-cofactor activity suggesting that the affinity of protein S for phospholipids is of less importance in the expression of APC-cofactor activity of protein S. We conclude that high affinity interactions of protein S with the membrane surface are essential for the direct anticoagulant activity of protein S and we suggest that inhibition of the prothrombinase and the tenase complex by protein S is a consequence of the occupation of the phospholipid surface by protein S molecules.

A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S.

To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in a plasma sample divided by the clotting time in the presence of a polyclonal antibody against human protein S (both after 1:1 dilution in protein S-C4BP deficient plasma). The mean protein S-dependent anticoagulant ratio (PSdAR) was 1.53+/-0.18 in 34 healthy controls, and was significantly lower in 16 heterozygous protein S deficient patients (PSdAR=1.15+/-0.09). In plasmas from patients under oral anticoagulant therapy the mean PSdAR was within the range of the control population (1.50+/-0.18). The mean total protein S antigen level in these plasmas was 58%, suggesting a higher specific APC-independent anticoagulant activity of protein S in these patients than in normals. This functional protein S assay can be used for the laboratory diagnosis of protein S deficiency, and to study the mechanism of the APC-independent anticoagulant activity in plasma.

Regulation of prothrombinase activity by protein S.

The independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (approximately 0.2 microM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd approximately 10(-8) M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and prothrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 x 10(8)/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.