Coated platelets and severe haemophilia A bleeding phenotype: Is there a connection?

INTRODUCTION: Coated platelets are a subpopulation of platelets that possess highly prothrombotic properties. Previous observational data suggest that bleeding phenotype in severe haemophilia A is associated with coated platelet levels. Haemophilia A patients with higher coated platelet levels may have a mild bleeding phenotype; those with lower levels may have a more severe bleeding phenotype. AIM: The aim of the study was to test the hypothesis that coated platelet levels are correlated with clinical bleeding phenotype. METHODS: This cross-sectional, observational study enrolled 20 severe haemophilia A patients, including 15 with severe and five with a mild bleeding phenotype, and a control group of 12 healthy volunteers. The haemophilia bleeding phenotype was determined by the patient's medical history and haemophilia treatment centre records. Blood was obtained from each patient by venipuncture and platelets were analysed by flow cytometry. RESULTS: Patients categorized as having a severe bleeding phenotype experienced a median eight bleeds per year compared to one bleed annually in the mild bleeding phenotype group. Both groups had similar total platelet counts and fibrinogen levels. There was no difference in coated platelet percentage between severe and mild bleeding phenotype (17 and 16% respectively), however, both groups had significantly lower % coated platelets compared to controls (44%, P < 0.0001). CONCLUSION: Coated platelet levels were not associated with bleeding phenotype in this study; however, these data may suggest coated platelet levels are lower in haemophilia patients relative to healthy volunteers.

Progressive improvement in wound healing with increased therapy in haemophilia B mice.

Previous work has shown that normalized haemostasis only at the time of an injury is not sufficient to promote optimal wound healing in haemophilia B (HB) mice. However, the duration of treatment required for optimal healing has not been established. The goal of these studies was to determine the effect of different durations of replacement or bypassing therapy [factor IX(FIX) or factor VIIa (FVIIa)] on wound healing parameters in a mouse model of HB. A dermal wound was placed on the back of HB mice. Animals were either untreated or pretreated and then subsequently treated for 3 days, 5 days, or 7 days with FIX or FVIIa. Wound area, time to wound healing, haematoma formation and iron deposition were measured. All treated animals showed shortened time to healing relative to untreated animals. Haematoma formation was prevented by treatment and bleeding into the wounds, measured by iron scores, was reduced by treatment. In addition, there was a progressive improvement in healing with 7 days of treatment more effective than 5 days which was more effective than 3 days. Replacement therapy with FIX had slightly shorter healing times than bypassing therapy with FVIIa. HB mice treated with FIX had slightly smaller wound area than untreated animals; by contrast, FVIIa-treated animals had much smaller wound areas that were close to the wound areas seen in wild-type animals. The data suggest that sustained therapy is required for normal wound healing.

Wound healing in haemophilia--breaking the vicious cycle.

Our group has been studying how haemostasis interacts with repair processes and also how to optimize treatment of bleeding disorders in a mouse model of haemophilia B. We have found that cutaneous wounds heal more slowly in haemophilic mice than in wild-type mice, and also exhibit histological abnormalities, even after closure of the skin defect. The haemophilic wounds showed reduced influx of inflammatory cells and increased angiogenesis. Even after surface closure, the haemophilic animals experienced repeated episodes of re-bleeding and progressive accumulation of iron in the wound bed and deeper tissues. A dose of replacement or bypassing therapy sufficient to establish initial haemostasis did not normalize wound healing. In fact, daily dosing for 7 days was required to normalize wound closure. Thus, normal healing requires adequate haemostatic function for an extended period of time. We have hypothesized that this is because angiogenesis during healing predisposes to bleeding, especially in the setting where haemostasis is impaired. Thus, normalizing haemostasis, until the process of angiogenesis has resolved, may be required to prevent re-bleeding and additional tissue damage.

Factor XI promotes hemostasis in factor IX-deficient mice.

Essentials Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model. FIX-deficient mice displayed a hemostatic defect and FXI-deficient mice were similar to wild type mice. Infusion of FXI or over-expression of FXI in FIX-deficient mice improved hemostasis. FXI may affect the phenotype of FIX-deficiency (hemophilia B). SUMMARY: Background In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI-deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI-deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model. Objectives To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B). Methods Wild-type mice and mice lacking either FIX (F9- ) or FXI (F11-/- ) were tested in the SVB model. The plasma levels of FXI in F11-/- mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection. Results F9- mice showed a significant defect in the SVB model, whereas F11-/- mice and wild-type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9- mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX-binding site also improved hemostasis in F9- mice. Conclusions Although we were unable to demonstrate a hemostatic defect in F11-/- mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9- mice through FIX-independent pathways.

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.

The tissue factor-factor VIIa complex: procoagulant activity, regulation, and multitasking.

Greater understanding of the cellular interactions associated with tissue factor (TF), activated factor (F) VII and TF-FVIIa complexes is likely to provide considerable clinical benefit. This article reviews current knowledge on the function and regulation of TF and its role in a range of biological processes, including hemostasis, thrombosis and inflammation.

Restoring hemostatic thrombin generation at the time of cutaneous wounding does not normalize healing in hemophilia B.

BACKGROUND: We recently reported that wound healing is abnormal in hemophilia B (HB) mice [1]. The wounds show abnormal histology: s.c. hematoma formation; delayed re-epithelialization; delayed macrophage influx; and an increase in wound site angiogenesis. OBJECTIVE: To test the hypothesis that restoring a hemostatic level of thrombin generation at the time of wounding would allow formation of an adequate platelet/fibrin plug and correct abnormalities of wound healing in HB. METHODS: We placed a 3-mm cutaneous wound on the back of each HB or wild-type (WT) mouse. Some HB mice were treated just prior to wounding with either human factor IX (FIX) or FVIIa in a dose sufficient to normalize bleeding in a tail bleed model. RESULTS: The average wound size over time in treated HB animals was intermediate between those in WT and untreated HB mice. However, the time to complete skin closure was not improved by treatment. Hematoma formation was decreased and macrophage influx began earlier in treated than in untreated HB animals. However, treated HB mice had evidence of ongoing low-level bleeding near the wound site, even after closure of the skin defect. Treatment also did not normalize the increased angiogenesis observed in HB mice. CONCLUSIONS: Restoring initial hemostasis can modulate some of the parameters of wound healing. However, an extended period of adequate hemostatic function is necessary to achieve normal healing, probably because the risk of hemorrhage is increased by vascular remodeling and angiogenesis during the healing process.

Tissue factor around dermal vessels has bound factor VII in the absence of injury.

BACKGROUND: 'Idling' or ongoing low-level activity of the tissue factor (TF) pathway is a postulated mechanism by which the coagulation process can become active without a lag period at sites of injury. OBJECTIVE: To determine whether TF around cutaneous vessels has bound factor VIIa in the absence of injury, and thus could participate in the idling process. METHODS: Immunostaining of mouse skin with antibodies against a 15-residue peptide from the sequence of mouse TF, and against the whole extracellular portion of TF. RESULTS: The whole TF antibody recognized TF in squamous epithelium and around vessels in the dermis. By contrast, the monospecific antibody only recognized TF in the squamous epithelium, but not around vessels. We also found that biotinylated, active site-inhibited FVIIa (FVIIai) bound to tissue sections in the same areas in which TF was recognized by the monospecific antibody (squamous epithelium), but did not bind around vessels. Molecular modeling revealed that FVIIa and FX binding to TF masked a significant part of the surface of the target peptide. CONCLUSIONS: In the aggregate, these data are most consistent with the interpretation that TF in perivascular sites has bound FVIIa, even in the absence of any injury. The presence of endogenously bound FVIIa prevents the subsequent binding of the monospecific antibody or exogenous FVIIai to perivascular TF.

Thrombin generation in vascular tissue.

BACKGROUND: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. METHODS AND RESULTS: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. CONCLUSION: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.

An activated factor VII variant with enhanced tissue factor-independent activity speeds wound healing in a mouse hemophilia B model.

UNLABELLED: Essentials Disorders of hemostasis can lead to delayed and defective wound healing. In hemophilia B (HB) mice, 7 days of Factor (F)IX or VIIa are needed to normalize wound healing. One dose of a highly active FVIIa variant (DVQ) restored normal wound closure time in HB mice. Coagulation factors with enhanced activity may acquire biological effects not due to hemostasis. SUMMARY: Introduction We have previously reported that hemophilia B (HB) mice have delayed healing of cutaneous wounds and alterations in wound histology. Administration of a single dose of either factor IX or recombinant activated FVII (rFVIIa) (NovoSeven) prior to wounding did not improve wound closure time or histology. The FVIIa analog DVQ (V158D, E296V and M298Q mutations) was designed to have higher tissue factor-independent activity than rVIIa. We hypothesized that a single dose of DVQ would be more effective in restoring wound healing in HB mice. Methods Cutaneous punch wounds were made on the backs of HB and wild-type mice, and the time to wound closure was monitored. HB mice were treated with a dose of rFVIIa (10 mg kg(-1) ) or DVQ (1 mg kg(-1) ) that corrected the tail bleeding time. Skin samples were taken at various time points after wounding, fixed, and stained, and the histology was examined. Results As previously reported, wound closure times in HB mice given one dose of rFVIIa were not improved over those in untreated HB mice. Surprisingly, healing times in HB mice treated with an equally hemostatic dose of DVQ were normalized to that in wild-type mice. However, DVQ did not correct all histologic abnormalities in HB mice. Conclusions As the doses of DVQ and rFVIIa were chosen to support comparable levels of hemostasis, our data suggest that the improved healing seen with DVQ is not solely attributable to its hemostatic activity. It is possible that the improved wound healing arises through the effect of DVQ on cell signaling mechanisms.

Thrombin generation and cell-dependent hypercoagulability in sickle cell disease.

Essentials Sickle cell disease is increasingly being recognized as a chronic hypercoagulable state. Thrombin generation is elevated in the whole blood, but not the plasma of sickle cell patients. Whole blood thrombin generation inversely correlates to erythrocyte phosphatidylserine exposure. Acquired protein S deficiency is likely explained by binding of protein S to sickle red cells. Click to hear Dr Hillery discuss coagulation and vascular pathologies in mouse models of sickle cell disease. SUMMARY: Introduction Sickle cell disease (SCD) is a hypercoagulable state with chronic activation of coagulation and an increased incidence of thromboembolic events. However, although plasma pre-thrombotic markers such as thrombin-anithrombin complexes and D-dimer are elevated, there is no consensus on whether global assays of thrombin generation in plasma are abnormal in patients with SCD. Based on our recent observation that normal red blood cells (RBCs) contribute to thrombin generation in whole blood, we hypothesized that the cellular components in blood (notably phosphatidylserine-expressing erythrocytes) contribute to enhanced thrombin generation in SCD. Methods Whole blood and plasma thrombin generation assays were performed on blood samples from 25 SCD patients in a non-crisis 'steady state' and 25 healthy race-matched controls. Results Whole blood thrombin generation was significantly elevated in SCD, whereas plasma thrombin generation was paradoxically reduced compared with controls. Surprisingly, whole blood and plasma thrombin generation were both negatively correlated with phosphatidylserine exposure on RBCs. Plasma thrombin generation in the presence of exogenous activated protein C or soluble thrombomodulin revealed deficiencies in the protein C/S anticoagulant pathway in SCD. These global changes were associated with significantly lower plasma protein S activity in SCD that correlated inversely with RBC phosphatidylserine exposure. Conclusion Increased RBC phosphatidylserine exposure in SCD is associated with acquired protein S deficiency. In addition, these data suggest a cellular contribution to thrombin generation in SCD (other than RBC phosphatidylserine exposure) that explains the elevated thrombin generation in whole blood.

The action of high-dose factor VIIa (FVIIa) in a cell-based model of hemostasis.

We have developed a cell-based model of hemostasis. This model suggests that the defect in hemophilia is specifically a failure of platelet-surface factor Xa (FXa) generation, leading to a failure of platelet surface thrombin generation. Activation of FX by FVIIa/tissue factor (TF) does not compensate for a lack of FXa activation on the platelet surface by the FVIIIa/FIXa complex. This is because plasma protease inhibitors prevent FXa from moving through the fluid phase from the TF-bearing cell to the platelet surface. We have previously proposed a platelet-dependent mechanism of action for high-dose factor VIIa (FVIIa; Novoseven, Novo Nordisk, Copenhagen, Denmark). Our data suggest that, when present at high levels, FVIIa binds to activated platelets and activates small amounts of FX independent of TF. This platelet-surface FXa can partially restore platelet-surface thrombin generation in hemophilia. Recently, van't Veer and colleagues reported results from an in vitro model in which coagulation reactions were initiated by relipidated TF. The authors concluded that high-dose FVIIa may exert a hemostatic effect in hemophilia by overcoming inhibition of FVIIa/TF activity by zymogen FVII. By contrast, we found that plasma levels of FVII did not slow thrombin generation in a model system initiated with cell-associated TF. This discrepancy highlights the potential differences between the studies of the coagulation reactions assembled on living cells compared to phospholipid vesicles. Our data suggest that in a cellular system high-dose FVIIa acts primarily by enhancing the rate of thrombin generation on platelet surfaces and not by overcoming inhibition by zymogen FVII of TF-dependent activation of FX.

Links between the immune and coagulation systems: how do "antiphospholipid antibodies" cause thrombosis?

Inflammation and immune activation have been associated with thrombosis in a number of settings. We have been interested in the question of how the presence of a type of autoantibody, so-called "antiphospholipid" antibody, leads to thrombosis. Several mechanisms have been proposed including modulation of tissue factor expression, enhancement of procoagulant binding to platelets, and interference with antithrombotic mechanisms. We developed a cell-based model of coagulation that, unlike current coagulation assays, reflects some of the in vivo activities of "antiphospholipid" antibodies. "Antiphospholipid" antibodies against the phospholipid-binding protein beta-2-glycoprotein-1 enhance thrombin generation in this model system, primarily by enhancing procoagulant reactions on tissue factor-bearing cells.

Recombinant human factor VIIa (rFVIIa) can activate factor FIX on activated platelets.

The studies reported here show that factor (F)VIIa can activate factor (F)IX on activated platelets in the absence of tissue factor. Both FIX and FIXa bind to the activated platelet surface with a K(d) of 8 nM and 2 nM, respectively. With factor (F)VIIIa, FIXa binds more tightly to platelets (K(d) 0.6 nM). At rFVIIa concentrations < 100 nm, no direct binding to the activated platelet surface can be detected with electrophoretic light scattering. However, in the presence of FIX, rFVIIa binding to platelets at concentrations as low as 10 nm rFVIIa can be detected. This is reflected by a decrease in the FIX K(d) from 8 to 1.6 nM. When rFVIIa is added to activated platelets in the presence of both FIX and FVIIIa, the K(d) for FIX decreases to 0.6, suggesting that rFVIIa activates FIX on the surface of activated platelets in the absence of tissue factor. The activation of FIX by FVIIa on activated platelets can also be demonstrated by a functional assay for FIXa. These data show that pharmacological doses of rFVIIa result in the direct activation of FIX by rFVIIa to form additional tenase complexes ultimately resulting in improved thrombin generation. These results may explain, at least in part, the mechanism of action of rFVIIa in hemorrhagic conditions seen in otherwise normal patients who develop an acquired coagulopathy due to trauma, surgery or a variety of other events in which rFVIIa has been found to be effective.

Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system.

Using a cell-based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0-200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV-deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell-based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin.

Role of the gamma-carboxyglutamic acid domain of activated factor X in the presence of calcium during inhibition by antithrombin-heparin.

BACKGROUND: Factor (F)Xa has 11 gamma-carboxylated glutamic acid (Gla) residues that are involved in calcium-dependent membrane binding. The serpin antithrombin (AT) is an important physiological regulator of FXa activity in an inhibition reaction that is enhanced by heparin. Recently, Rezaie showed that calcium further enhanced the heparin-catalyzed AT inhibition of FXa by promoting 'ternary complex' formation, and these results showed a role for the gamma-carboxyl-glutamate (Gla)-domain of FXa. OBJECTIVES: In this study, we used recombinant FXa mutants to assess the role of individual Gla residues in augmenting or antagonizing the AT-heparin inhibition reaction in the presence of calcium. RESULTS AND CONCLUSIONS: In the absence of heparin, AT inhibition of plasma and the recombinant FXas were essentially equivalent. Similar to plasma-derived FXa, calcium increased about 3-fold the inhibition rate of wild-type recombinant FXa by AT-heparin over that in the presence of EDTA. Interestingly, three different effects were found with the recombinant FXa Gla-mutants for AT-heparin inhibition: (i) Gla-->Asp 14 and 29 were enhanced without calcium; (ii) Gla-->Asp 16 and 26 were not enhanced by calcium; and (iii) Gla-->Asp 19 was essentially the same as wild-type recombinant FXa. These results support a theory that mutating individual Gla residues in FXa alters the calcium-induced conformational changes in the Gla region and affects the antithrombin-heparin inhibition reaction.

Microplate coagulation assays.

This report describes the development of microplate-based blood coagulation assays. The assays require a kinetic microplate reader to follow changes in absorbance at 405 nm caused by the coagulating plasma. Procedures for performing prothrombin time and activated partial thromboplastin time tests are described with intra- and inter-assay variability of a few percentage points. The prothrombin time of normal plasma was 64.5 +/- 3.6 s, and the activated partial thromboplastin time was 69.8 +/- 3.2 s. Clotting times were prolonged when normal plasma was mixed with plasmas deficient in particular coagulation factors, as expected. These assays take advantage of the microplate format (small sample size and multiple simultaneous assays) and can be customized for specific purposes, such as quantifying purified factor IX or assessing protein C activity in plasma.

A cell-based model of hemostasis.

Based on our work and that of many other workers, we have developed a model of coagulation in vivo. Many workers have demonstrated mechanisms by which cells can influence the coagulation process. Nonetheless, the prevailing view of hemostasis remains that the protein coagulation factors direct and control the process with cells serving primarily to provide a phosphatidylserine containing surface on which the procoagulant complexes are assembled. By contrast, we propose a model in which coagulation is regulated by properties of cell surfaces. This model emphasizes the importance of specific cellular receptors for the coagulation proteins. Thus, cells with similar phosphatidylserine content can play very different roles in hemostasis depending on their complement of surface receptors. We propose that coagulation occurs not as a "cascade", but in three overlapping stages: 1) initiation, which occurs on a tissue factor bearing cell; 2) amplification, in which platelets and cofactors are activated to set the stage for large scale thrombin generation; and 3) propagation, in which large amounts of thrombin are generated on the platelet surface. This cell based model explains some aspects of hemostasis that a protein-centric model does not.