Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4.

Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma cholesterol and its potential pathophysiologic and clinical relevance.

Diagnosis of mild platelet function disorders. Reliability and usefulness of light transmission platelet aggregation and serotonin secretion assays.

Light transmission platelet aggregation (PA), adapted to measure platelet secretion (PS), is the reference test for diagnosing platelet functional disorders (PFD). Problems with these assays include lack of standardisation, unknown reproducibility and lack of universally accepted diagnostic criteria. We addressed these issues in patients with inherited mucocutaneous bleeding (MCB). Normal and abnormal PA tests in 213 patients were reproducible in 93.3% and 90.4% of the cases, respectively. Mean intra-subject coefficient of variation for PA with strong agonists were <9% and mean intra-class correlation coefficient for weak agonists were >0.86 (P < 0.0001). Concomitant impaired PA with 10 micromol/l-adrenaline and 4 micromol/l-ADP was observed in 13.7% of the controls. This combination was not considered per se a criterion for PFD. PA with adrenaline > or = 42% or irreversible aggregation with 4 micromol/l ADP had 93% and 95% Negative Predictive Value for diagnosing PFD, respectively. PA defects were consistently associated with abnormal PS. In contrast, 14.3% of patients with MCB had isolated PS. Thus, standardized PA/PS assays are highly reproducible and concordant in normal and patient populations. Normal PA with adrenaline and low ADP concentration robustly predict a normal PA. Simultaneous PA/PS assays enable the diagnosis of isolated PS defects. This study confirmed that hereditary PA-PS defects are highly prevalent.

Procarboxypeptidase U (TAFI) and the Thr325Ile proCPU polymorphism in patients with hereditary mucocutaneous hemorrhages.

BACKGROUND: Patients with hereditary mucocutaneous bleeding are difficult to diagnose and many of them fulfill the category of bleeders of unknown cause (BUC). The pathogenic role of hyperfibrinolysis has received little attention, despite the successful use of antifibrinolytic drugs in treating many of these patients. Theoretically, decreased plasma procarboxypeptidase U (proCPU) levels or lower carboxypeptidase U (CPU) stability would result in higher fibrinolytic activity and bleeding tendency. METHODS: We analyzed plasma proCPU and the distribution of the Thr325Ile proCPU polymorphism in 193 patients with mucocutaneous bleeding of whom 116 were bleeders of unknown cause (BUC), and in 143 healthy, age and sex-matched controls. RESULTS: ProCPU concentration was higher in women than in men, increased with age, and was significantly correlated with clot lysis time, platelet count, APTT, and PT. However, proCPU levels were unexpectedly higher in patients than in controls (968+/-134 vs. 923+/-147 U/L, p=0.004). The allele distribution of the Thr325Ile proCPU polymorphism was similar in both groups, with a low percentage of homozygous Ile/Ile. CONCLUSIONS: Our results indicate that the proCPU system is not of major importance in the bleeding pathogenesis of these patients. The higher proCPU levels in the patients may even modestly counteract the bleeding tendency.

Tissue factor storage, synthesis and function in normal and activated human platelets.

The source and significance of blood-borne tissue factor (TF) are controversial. The presence of TF in platelets was initially attributed to transfer of the protein from other cells (e.g., monocytes) and/or TF-bearing microparticles. Recently, TF-mRNA, neo-synthesis of the protein and TF-dependent procoagulant activity (PCA) have been reported in human platelets. The storage of "encrypted", potentially active TF in circulating, non-stimulated platelets remains debatable. One report strongly suggests that the starting of platelet PCA depends on de novo TF synthesis induced by platelet activation, whereas others provide persuasive evidence that platelets circulate with preformed TF, readily functional upon demand. These findings may have an impact on our current ideas of physiological hemostasis and thrombus formation. In fact, platelets would lead not only the formation of the primary plug, but in this microenvironment they would also contribute to the triggering of thrombin generation, fibrin deposition, clot consolidation and initial protection from fibrinolysis. Much research is needed to validate this platelet-based hemostasis model.

Human platelets synthesize and express functional tissue factor.

The source and significance of bloodborne tissue factor (TF) are controversial. TF mRNA, protein, and TF-dependent procoagulant activity (PCA) have been detected in human platelets, but direct evidence of TF synthesis is missing. Nonstimulated monocyte-free platelets from most patients expressed TF mRNA, which was enhanced or induced in all of them after platelet activation. Immunoprecipitation assays revealed TF protein (mainly of a molecular weight [Mr] of approximately 47 kDa, with other bands of approximately 35 and approximately 60 kDa) in nonstimulated platelet membranes, which also increased after activation. This enhancement was concomitant with TF translocation to the plasma membrane, as demonstrated by immunofluorescence-confocal microscopy and biotinylation of membrane proteins. Platelet PCA, assessed by factor Xa (FXa) generation, was induced after activation and was inhibited by 48% and 76% with anti-TF and anti-FVIIa, respectively, but not by intrinsic pathway inhibitors. Platelets incorporated [(35)S]-methionine into TF proteins with Mr of approximately 47 kDa, approximately 35 kDa, and approximately 60 kDa, more intensely after activation. Puromycin but not actinomycin D or DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) inhibited TF neosynthesis. Thus, human platelets not only assemble the clotting reactions on their membrane, but also supply their own TF for thrombin generation in a timely and spatially circumscribed process. These observations simplify, unify, and provide a more coherent formulation of the current cell-based model of hemostasis.

Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state.

The risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), affecting both venous and arterial vessels. Activated platelets are known to participate in thrombus formation and growth. A general feature of activated cells is the shedding of microparticles (MP) which support coagulation by exposure of negatively charged phospholipids and possibly tissue factor (TF). In this work we characterized circulating MP in patients with SLE and their relationship with a procoagulant state. Thirty patients with SLE (aged 15-72 years, mean age 38 years) and 20 healthy controls (aged 22-54 years, mean age 34 years) were studied; patients fulfilled 4 revised criteria for SLE. The number and cellular source of circulating MP were determined by flow cytometry using double labeling with specific monoclonal antibodies and annexin V. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and TF; under these conditions the generation of thrombin depended directly on the number of MP present in plasma. Thrombin anti-thrombin (TAT) and plasmin-antiplasmin (PAP) complexes were measured by ELISA. Compared to the controls, circulating MP were significantly elevated in the patient group (1218 +/- 136 vs 653 +/- 74 x 10(3)/ml plasma, p: 0.0007). In both groups, most of these MP were of platelet origin (927 +/- 131 vs 517 +/- 72 x 10(3)/ml plasma, p:0.009 ). ETP was higher among patients as compared to the controls (804 +/- 64 vs 631 +/- 37 nM thrombin, p: 0.025). Plasma levels ofTAT in patients and controls were 3.4 +/- 0.8 and 1.4 +/- 0.5 microg/L, respectively (p:0.04), and of PAP complexes were 62.5 +/- 14 and 24.05 +/- 2.5 microg/ml, respectively (p: 0.014). The number of platelet-derived MP correlated significantly with thrombin generation (r: 0.42; p: 0.038) and TAT levels (r: 0.40; p: 0.035). We did not find an association of circulating MP with disease activity nor with the presence of antiphospholipid antibodies. The increased number of circulating platelet-derived microparticles and their association with high ETP and activation of the coagulation system suggest that these microparticles play an important role in the pathogenesis of the prothrombotic state in SLE patients.