Microparticle-linked tissue factor activity and increased thrombin activity play a potential role in fibrinolysis failure in ST-segment elevation myocardial infarction.

Fibrinolysis for acute ST-segment elevation MI achieves early recanalisation of the infarct artery in approximately 60% of cases. The aim of the study was to determine whether failure to achieve recanalisation was associated with differences in haemostasis biomarkers compared to patients with successful fibrinolysis. Fourty-three patients were prospectively enrolled in a case-control study. All patients had received tenecteplase (TNK-tPA) together with aspirin (500 mg) and heparin (5,000 IU). Emergency angiography within 90 minutes of bolus TNK-tPA identified 13 TIMI 0-2 patients (cases) and 30 TIMI 3 patients (controls). Blood samples were collected before angiography to determine tissue factor activity associated with microparticles (TF-MP); soluble platelet glycoprotein V (sGPV) and thrombin-antithrombin complexes (TAT) as markers of thrombin generation; tissue plasminogen activator (endogenous tPA+ TNK-tPA), plasminogen activator inhibitor (PAI-1) and plasmin-antiplasmin complexes (PAP) as markers of plasmin generation. The baseline characteristics of the two patients' groups were similar with respect to sex, age, and risks factors. Cases differed from controls by higher TF-MP levels (1.9 [1-13] vs. 1 [0.6-1.3] pM), sGPV (67 [51-126] vs. (48 [39-72] ng/ml), p = 0.039 and TAT (10 [4-37.5] vs. 4 [2.9-7.2] ng/ml), p = 0.035. TAT correlated with TF-MP (r = 0.51, p = 0.0064) and sGPV (r = 0.51, p = 0.0018). No significant difference was observed in tPA or PAI-1 levels. PAP were lower in cases (18.83 [14.83-40.43] mug/ml) than in controls (35.83 [27.9-43.94] mug/ml), p = 0.045. In conclusion, fibrinolysis failure in AMI is characterised by a higher procoagulant state associated with TF-MP and a lower plasmin generation.

Leukocyte activation: the link between inflammation and coagulation during heatstroke. A study of patients during the 2003 heat wave in Paris.

OBJECTIVE: The mechanisms linking severe inflammation and coagulation during heatstroke are poorly understood. Here, we examined the roles of the tissue factor pathway, leukocyte activation, and mediators of innate immunity in patients admitted to an intensive care unit for heatstroke during an intense heat wave in Paris. DESIGN: Retrospective observational study. SETTING: Intensive care unit of a university medical center. PATIENTS: Eighteen critically ill severe patients with heatstroke were enrolled in the study and 14 age-matched patients with severe sepsis as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: High circulating levels of some inflammation and stress mediators (interleukin-6, -8, C5a, interleukin-1 receptor antagonist, heat shock protein 60 and 70) were observed. Blood leukocyte activation was shown by beta2 integrin up-regulation, L-selectin down-regulation, and strong production of reactive oxygen species and interleukin-8 ex vivo. High levels of circulating promatrix metalloproteinase-9 were detected in all the patients studied, and its active form was present in two patients. Overt disseminated intravascular coagulation according to the International Society of Thrombosis and Hemostasis score was present in five patients. Whole-blood tissue factor was present in all the patients and part of this activity was associated with microparticles in five patients. The degrees of inflammation and disseminated intravascular coagulation are correlated with clinical severity. CONCLUSIONS: These results suggest that neutrophil activation plays a key role in the acute activation of coagulation observed during severe heatstroke, despite a rapid and sustained antiinflammatory response. The comparison with a group of patients with severe sepsis suggests some common mechanisms, but more intense responses during heatstroke.

Prothrombotic markers and early spontaneous recanalization in ST-segment elevation myocardial infarction.

We tested the hypothesis that selected prothrombotic biomarkers might be associated with early spontaneous coronary recanalization in patients with ST-segment elevation acute myocardial infarction (STEMI). We prospectively enrolled 123 patients with STEMI including 53 patients with spontaneous coronary recanalization (cases) and 70 patients with persistent occlusion (controls) at the time of emergent coronary angiography and before angioplasty. All had received aspirin and heparin. Blood samples were collected immediately before angioplasty to measure soluble P-selectin, circulating microparticles originating from platelets (PMPs), granulocytes (GMPs), endothelial cells (EMPs); tissue factor-associated MP (TF-MP); soluble platelet glycoprotein V (sGPV) and prothrombin F1 + 2; tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI-1) and plasmin-antiplasmin (PAP). A sub-group of 70 patients (35 cases, 35 controls) was available for flow cytometry analysis of platelet P-selectin and activated GPIIb-IIIa. Baseline clinical characteristics did not differ between groups except for more frequent hypertension and dyslipidemia in controls. Platelet activation markers and PMP did not differ between the two groups. Controls had higher numbers of EMPs and GMPs compared to cases, but the difference was no longer significant when corrected for risk factors. Controls differed from cases by higher plasma levels of sGPV [64 (47-84) ng/ml vs. 53 (44-63) ng/ml] and PAP [114(65-225) ng/ml vs. 88 (51-147) ng/ml]. The difference persisted after adjustment for risks factors (p = 0.031 and 0.037, respectively). Persistent occlusion of the infarct related artery is associated with some markers related to higher thrombin (sGPV) and plasmin (PAP) production but is not associated with markers of platelet activation.

Hypercoagulability after partial liver resection.

One concern of living donor liver transplantation remains the risk of morbidity and/or mortality for the donors, including the risk of postoperative thrombosis. We studied the coagulation changes after partial liver resection in l2 living donors and eight patients with non-malignant hepatic tumors (controls) and searched for potential predictive markers of thrombotic complications. Thrombosis (pulmonary embolism and portal vein thrombosis) developed in two donors and two controls. In donors and controls, we observed an early postoperative decrease in coagulation inhibitors protein C and antithrombin together with an increase in factor VIII and von Willebrand factor, which both persisted when prothrombin time had returned to normal. Dysregulation in the haemostatic system was confirmed by increased prothrombotic markers, with a 10- to 30-fold increase in thrombin-antithrombin complexes and moderate increase(1.5- to 2.0-fold) in sP-Selectin. No difference between donors and controls was observed and the data were pooled for comparison of patients with (n = 4) versus without (n = 16) thrombosis. Thrombin-antithrombin complexes were significantly higher in the thrombosis group, on day 1 (28.8 vs. 13.5 microg/l, p = 0.027) and day 2 (52.3 vs. 9.3 microg/l, p = 0.013). sP-selectin was also significantly higher in the thrombosis group on day 2 (103 vs. 53 ng/ml, p = 0.044) and day 4 (116 vs. 58 ng/ml, p = 0.026) after surgery. Our study indicates that improvement of thromboprophylaxis in partial liver resection is needed. It also suggests that thrombin-antithrombin complexes and sP-selectin could serve as early biological predictors of thrombotic complications in the post-operative period.

A new plastic collection tube made of polyethylene terephtalate is suitable for monitoring traditional anticoagulant therapy (oral anticoagulant, unfractionated heparin, and low molecular weight heparin).

To improve the safety of blood collection, plastic tubes have been developed but various interactions with the coagulation system and/or antithrombotic drugs were reported with the first generation of such tubes. The aim of this multicentre study was to compare hemostasis test results measured in evacuated plastic tubes made of polyethylene terephtalate (VenoSafe, Terumo Europe) and in siliconized glass tubes containing the same citrate concentration (0.129 M). In addition, the impact of aging of the plastic tube was investigated by collecting blood samples in tubes at 8 months and at 1 month before expiry. Blood was drawn in 3 centres from untreated patients (n=269), patients on oral anticoagulant treatment (OAT, n=221), and patients treated with either unfractionated heparin (UFH, n=73) or a low molecular weight derivative (LMWH, n=48). Prothrombin time (PT) or INR, activated partial thromboplastin time (APTT) and anti-FXa activity were locally performed, when applicable. In untreated patients and in patients on OAT, PT and APTT values were found statistically shorter (p<0.05) when evaluated in plastic tubes than in glass tubes, except when PT was evaluated using a human thromboplastin. Surprisingly, significantly longer APTT and higher anti-FXa activities were obtained when blood from patients on UFH was drawn in plastic than in glass tubes. However, none of the differences had any clinical relevance (Bland-Altman analysis). In patients on anticoagulant treatment, there was no effect of aging of the plastic tubes. These results suggest that the plastic tube VenoSafe is suitable for coagulation testing both in untreated subjects and more interestingly in patients on traditional anticoagulant therapy during the whole shelf life indicated by the manufacturer.

Enhanced shear-induced platelet aggregation in patients who experience subacute stent thrombosis: a case-control study.

OBJECTIVES: The goal of this study was to identify differences in shear-induced platelet aggregation (SIPA) between patients who did or did not experience subacute stent thrombosis (SAT). BACKGROUND: Despite dual antiplatelet therapy, SAT after coronary stenting occurs in approximately 1% of patients. There is no accepted platelet function test to identify patients at risk. METHODS: We analyzed platelet aggregation in 10 patients who had experienced SAT (cases), 22 stented patients without SAT (controls), and 17 healthy volunteers (normals). All patients except normals were treated with both aspirin and clopidogrel. RESULTS: Shear-induced platelet aggregation was higher in cases than in controls at both shear rates of 200 s(-1) (40.9 +/- 12.2% vs. 18.2 +/- 18%, p = 0.013) and 4,000 s(-1) (57.4 +/- 16.4% vs. 23.4 +/- 21.2%, p = 0.009). Moreover, SIPA in cases was significantly higher than in normals both at 200 s(-1) (p = 0.013) and 4,000(-1) (p = 0.009). CONCLUSIONS: Shear-induced platelet aggregation is increased in patients experiencing SAT compared with controls receiving dual antiplatelet therapy and to normals receiving no antiplatelet therapy, which suggests increased intrinsic patient-related platelet reactivity in patients with SAT. The predictive value of SIPA for SAT requires prospective investigation.

Association of the -92C/G and 807C/T polymorphisms of the alpha2 subunit gene with human platelets alpha2beta1 receptor density.

OBJECTIVE: Platelet adhesion to the subendothelial tissue via the collagen receptor alpha2beta1 is a crucial event in vascular biology. Although evidence has been provided that the number of platelets alpha2beta1 copies is genetically determined, the molecular change primary responsible has not been yet elucidated. The aim of our present study was to investigate the effect of combined polymorphisms within both regulatory (-52C/T and -92C/G) and coding regions (807C/T and 1648A/G) of the alpha2 subunit gene on human platelets alpha2beta1 receptor density and/or susceptibility to coronary artery disease (CAD). METHODS AND RESULTS: Among 254 cardiac surgery patients, no evidence was found for an association between the alpha2 subunit gene polymorphisms and CAD. In contrast, in a subgroup of 113 patients, we observed a significant association between all polymorphisms except -52C/T and alpha2beta1 receptor level. Furthermore, when 3 groups of patients were defined according to the tertiles of platelets alpha2beta1 copies, the -92C/807T haplotype was more frequent in the group of patients with high alpha2beta1 receptor level. CONCLUSIONS: These results suggest that an individual effect of each polymorphism located either in the coding or promoter sequence of the alpha2 gene may act in combination to modulate variations in platelets alpha2beta1 receptor density.

Plasma matrix metalloproteinase-9 as a marker of blood stasis in varicose veins.

BACKGROUND: Possible intermediate circulating markers linking blood stasis to vein remodeling were explored in patients with varicose veins in the lower limbs. METHODS AND RESULTS: Blood was sampled at rest (supine position) and after a stasis of 30 minutes both in the varicose vein (limbs hanging down) and in the brachial vein (arm hanging down) as a paired control. Several endothelial and leukocyte markers were measured in plasma with the use of ELISA kits. Angiotensin-converting enzyme activity was determined by use of a specific substrate. Matrix metalloproteinases (MMPs) 9 and 2 were evaluated with the use of gelatin zymography. No markers were significantly modified after 30 minutes of blood stasis in the brachial vein. After 30 minutes of blood stasis in the varicose vein, oxygen partial pressure decreased (P<0.01). Although thrombomodulin, von Willebrand factor, vascular endothelial growth factor, and MMP-2 were not modified in these conditions, the proteins released by proteolysis from the endothelial membrane intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and angiotensin-converting enzyme were increased (P<0.01). After blood stasis in varicose veins, the leukocyte markers lactoferrin, myeloperoxidase, and interleukin-8 were not modified, whereas L-selectin shed from leukocytes increased (P<0.05), and a major increase in pro-MMP-9, which is released from tertiary granules during polymorphonuclear activation, was observed (P=0.0001). CONCLUSIONS: The marked increase in plasma pro-MMP-9 activity provides evidence of polymorphonuclear activation and granule release in the varicose vein in response to postural blood stasis. Similarly, detection in the plasma of membrane proteins shed from the endothelium or leukocytes provides evidence of pericellular proteolysis.

The serpin protease-nexin 1 is present in rat aortic smooth muscle cells and is upregulated in L-NAME hypertensive rats.

OBJECTIVE: Protease-nexin 1 (PN-1) belongs to the serpin superfamily and behaves as a specific thrombin inhibitor in the pericellular environment. Little is known about PN-1 expression and its regulation in the vascular system. In this study, we examined the expression of functionally active PN-1 in vitro in rat aortic smooth muscle cells and in vivo in rat arterial media and its regulation in hypertensive rats. METHODS AND RESULTS: The vascular PN-1 formed specific covalent complexes with thrombin involving the catalytic site of the protease, and heparin increased the formation of these complexes. We also demonstrated PN-1 in rat arterial media by immunohistochemical staining. Moreover, we examined in vivo vascular expression of PN-1 in a model of chronic hypertension induced by long-term administration of N(G)-nitro-L-arginine methyl ester (L-NAME). Marked increases in PN-1 mRNA (3-fold) and protein (2-fold) were observed after 2 months of hypertension. Increased expression of PN-1 in the vascular wall was associated with an increase in the formation of complexes between radiolabeled-thrombin and PN-1, indicating that PN-1 was functional. CONCLUSIONS: PN-1 may thus participate in the mechanisms that regulate thrombin activity in the vessel wall.

Prothrombin Saint-Denis: a natural variant with a point mutation resulting in Asp to Glu substitution at position 552 in prothrombin.

A new prothrombin variant, with a point mutation at nucleotide 20 029 resulting in Asp 552 to Glu substitution (prothrombin numbering), has been identified in a male newborn. Plasma prothrombin level was <3%, 16% and 60% when measured by clotting, chromogenic and immunological assays respectively. The substitution did not affect the rate of prothrombin conversion to thrombin but altered thrombin activity. Amino acid 552 has been reported to be involved in the allosteric transition, which is induced by sodium binding to thrombin. This is the first known amino acid substitution at this site to result in dysprothrombinaemia.

Renewal of mural thrombus releases plasma markers and is involved in aortic abdominal aneurysm evolution.

Human abdominal aortic aneurysm (AAA) expansion has been linked to the presence of a mural thrombus. Here we explored the mechanism of the continual luminal renewal of this thrombus and its ability to release biological markers potentially detectable in plasma. We also explored the ability of platelet inhibition to pacify the thrombus and to limit aneurysm progression in an experimental model. Blood samples and mural thrombi were collected in 20 AAA patients. In parallel, segments of sodium dodecyl sulfate-decellularized guinea pig aorta were xenografted onto the abdominal aorta of 30 rats to induce aneurysms. Fifteen rats received abciximab treatment and fifteen received irrelevant immunoglobulins. Procoagulant activity and platelet activation markers (microparticles, sP-selectin, sGPV, sCD40L) were increased threefold to fivefold in eluates from the luminal thrombus layer compared to other layers. All these markers were increased twofold to fivefold in patients' plasma compared to matched controls (P < 0.005). In the rat model, abciximab reduced both thrombus area and aneurysmal enlargement (P < 0.05). Platelet aggregation is probably responsible for the renewal of the thrombus in AAA. The luminal thrombus released markers of platelet activation that could easily be detected in plasma. Platelet inhibition limited aortic aneurysm expansion in a rat model, providing new therapeutic perspectives in the prevention of AAA enlargement.

Glycoprotein Ib-mediated platelet activation. A signalling pathway triggered by thrombin.

Platelet activation by thrombin plays a major role in the development of haemostasis and thrombosis. Thrombin activates human platelets by cleaving the N-terminal region of G-protein-coupled protease-activated receptors (PARs). On the other hand, the platelet membrane glycoprotein GPIb acts as a thrombin-binding site and promotes platelet activation by low thrombin concentrations. We present here new evidence in favour of a thrombin receptor function for GPIb. We have selected conditions in which thrombin-GPIb interactions were enhanced by thrombin immobilization. Activation was studied independently of PAR cleavage by using active-site-blocked thrombin. We show that immobilized, proteolytically inactive thrombin induces platelet adhesion and spreading, dense granule secretion and integrin alphaIIbbeta3-dependent platelet-platelet interactions. The pathway must be dependent on GPIb because it is deficient in platelets from a patient with Bernard Soulier syndrome and inhibited by a monoclonal antibody to GPIb (SZ2) or by an excess of glycocalicin. Secreted ADP plays a major role in GPIb-dependent thrombin-induced platelet activation which is, in addition, regulated by cAMP concentration. Thrombin-induced GPIb-dependent platelet activation leads to tyrosyl phosphorylation of several proteins. Inhibition of platelet-platelet interactions and protein tyrosine phosphorylations by inhibitors of phosphatidylinositol 3-kinases and protein kinase C implies that activation of the latter are important steps of the GPIb-coupled signalling pathway triggered by thrombin.

Red blood cells from patients with homozygous sickle cell disease provide a catalytic surface for factor Va inactivation by activated protein C.

The structure of red blood cell (RBC) membranes in homozygous sickle cell disease (SCD) is significantly disturbed, with an increased exposure of aminophospholipids (phosphatidylserine and phosphatidylethanolamine) at the outer surface, responsible for a procoagulant activity of SS RBCs. Aminophospholipids are known not only to promote procoagulant reactions, but also to support inhibition of blood coagulation by the protein C system. The aim of the present study was to examine whether SS RBCs could serve as a catalytic surface for the inactivation of factor Va by activated protein C (APC). Venous blood was obtained from 19 consecutive SS patients and 13 controls (AA). In all SS patients, the amount of phosphatidylserine exposed at the outer surface of RBCs was increased compared with controls, as demonstrated by a prothrombinase assay. In addition, SS RBCs significantly (P < 0.0001) increased the rate of FVa inactivation by APC: the mean values (and ranges) of the factor Va inactivation rates were 30 (0-57) vs 9.5 (0-32) mmol Vai/min/mol APC for SS RBCs and normal RBCs respectively. Our results indicate that SS RBCs provide a catalytic surface for the negative control of blood coagulation, which may partially control the procoagulant activity of these cells.

Protease nexin-1: a cellular serpin down-regulated by thrombin in rat aortic smooth muscle cells.

Protease nexin-1 (PN-1), a potent inhibitor of serine proteases, is present in vascular cells and forms complexes with thrombin, plasminogen activators, and plasmin. We examined the effect of thrombin on PN-1 expression by rat aortic smooth muscle cells (RASMCs). PN-1 expression was determined by measuring protein and mRNA levels, using respectively immunoblotting and semi-quantitative reverse transcriptase polymerase chain reaction (PCR). Thrombin down-regulated PN-1 expression in a dose- and time-dependent manner. This effect was mediated via the interaction of thrombin with its receptor protease activated receptor (PAR-1) since the peptide thrombin receptor activating peptide (TRAP) reduced PN-1 expression. PN-1 secreted by smooth muscle cells remained essentially associated to cell-surface glycosaminoglycans and was released from the cell surface by heparin. A lower amount of PN-1 was released by heparin from TRAP-stimulated versus unstimulated cells and correlated with a decreased capacity to inhibit thrombin. In addition, the ability to generate peri-cellular plasmin was increased in cells with a low PN-1 expression. Pre-treatment of smooth muscle cells with cycloheximide abolished the reduction of PN-1 expression by thrombin. Furthermore, conditioned media from thrombin-treated cells reproduced the effect of thrombin, suggesting that thrombin acted via the induction of auto/paracrine mediator(s). We observed that fibroblast growth factor-2 (FGF-2)-neutralizing antibodies abolished thrombin effect whereas FGF-2 reproduced it, indicating that FGF-2 is one of the involved mediator. Together, these results indicate that (i) PN-1 modulates the activity of endogenous and exogenous serine proteases in RASMCs, (ii) thrombin down-regulates PN-1 expression and thus may increase its own activity on cells.

Protease nexin-1 inhibits plasminogen activation-induced apoptosis of adherent cells.

Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K(m) = 46 nm). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.

A paradoxical pro-apoptotic effect of thrombin on smooth muscle cells.

Whereas thrombin (below 10 nM) is a potent mitogen, recent studies report that exposure to higher doses of thrombin could lead to apoptosis of neurons and tumor cells. Our results show that prolonged exposure (> or = 24 h) to thrombin (50-100 nM) exerts a pro-apoptotic effect on cultured vascular smooth muscle cells (VSMCs). This phenomenon depends on thrombin serine-protease activity but is independent of PAR-1 and -4 activation and subsequent signaling. The parallel occurrence of cell retraction and cleavage of fibronectin suggests that thrombin-induced apoptosis is consecutive to pericellular proteolysis. These data point to a new potential action of thrombin in the cardiovascular system.

Hemostasis imbalance in experimental hypertension.

BACKGROUND: The rat model of chronic intoxication by N(G) -nitro-L-arginine methyl ester (L-NAME) induces severe systemic arterial hypertension and progressive ischemic lesions in the central nervous system and kidneys. We investigated the possible molecular basis of these thrombotic events. METHODS AND RESULTS: Administration of L-NAME increased plasma markers of thrombin generation, thrombin-antithrombin complexes, and soluble glycoprotein V, measured by specific ELISA. Thrombin generation in vivo was associated with ex vivo platelet desensitization to adenosine 5'-diphosphate and collagen-induced aggregation. In the aortic layers and renal arterioles, tissue factor mRNA (semi-quantitative RT-PCR) and activity (coagulation assay) were increased. In contrast, tissue factor activity was not modified in glomeruli. In parallel, an impairment of the fibrinolytic system was demonstrated by an increase in plasma levels and arterial secretion of plasminogen activator inhibitor-1. In the arterial wall, plasminogen activator inhibtor-1 mRNA was significantly increased. Moreover, antifibrinolytic activity, studied by fibrin reverse zymography, was increased whereas all tissue-plasminogen activator activity secreted by the hypertensive arterial wall was detected as complexes with its specific inhibitor. In animals treated with the angiotensin-converting enzyme (ACE) inhibitor Zofenil, all of these parameters remained at control levels. CONCLUSIONS: These results indicate that chronic blockade of nitric oxide production in rats results in enhancement of blood markers of thrombin generation associated with tissue factor induction and impairment of fibrinolysis in the vascular wall, which may contribute to the thrombotic complications associated with hypertension.