Platelets significantly modify procoagulant activities in haemophilia A.

Haemophilia A replacement therapy is dosed according to patient's weight and plasma FVIII activity (FVIII:C). The FVIII interacts with platelet membrane but limited data on the impact of platelet procoagulant activity (PCA) are available in haemophilia A. Our aim was to characterize individual PCA in vitro in 20 adult haemophilia A patients at various FVIII:C levels. We detected thrombin generation in platelet-poor (PPP) and platelet-rich plasma (PRP) using: (i) calibrated automated thrombography (CAT) triggered with tissue factor, (ii) adhesion-induced PCA upon collagen and (iii) annexin V binding, expression of P-selectin and active glycoprotein (GP) IIbIIIa on platelets after stimulation of GPVI with collagen-related peptide. The FVIII:C levels varied between <1% and 37%. Thrombin generation was individual and strongly enforced by platelets and associated within the three methods. Range of thrombin generation was maximal (up to 30-fold) at FVIII:C levels 1-5%, underlining the impact of platelets in the presence of traces of replacement therapy. At FVIII:C > 5% platelet contribution in the variance faded. Platelet PCA and P-selectin exposure lead to a fivefold variation. Intriguingly, at FVIII:C < 1% thrombin generation in PPP associated negatively with platelet GPVI activation, suggestive of a regulatory interplay between plasma and platelets. In haemophilia A, the variability in thrombin generation is partially related to plasma FVIII:C, but mainly dependent on platelet procoagulant capacity. Annexin V binding and PCA in response to activation by collagen receptors contribute to this variability. In all, platelet PCA at least following collagen interaction significantly impacts thrombin generation in haemophilia A.

Procoagulant activity on platelets adhered to collagen or plasma clot.

In a new 2-stage assay of platelet procoagulant activity (PCA), we first subjected gel-filtered platelets to adhesion on collagen (as a model of primary hemostasis) or plasma clots (as a model of preformed thrombus) for 30 minutes, and then the adherent platelets were supplemented with pooled, reptilase-treated, diluted plasma. Defibrinated plasma provided coagulation factors for assembly on platelet membranes without uncontrolled binding of thrombin to fibrin(ogen). Platelet adhesion to both surfaces showed modest individual variation, which increased at platelet densities that allowed aggregation. However, adhesion-induced PCA varied individually and surface-independently >3-fold, suggesting a uniform platelet procoagulant mechanism. Permanently adhered platelets showed markedly enhanced PCA when compared with the platelet pool in suspension, even after strong activation. The rate of thrombin generation induced by clot-adherent platelets was markedly faster than on collagen-adherent platelets during the initial phase of coagulation, whereas collagen-induced PCA proceeded slowly, strongly promoted by tissue thromboplastin. Therefore at 10 minutes, after adjustment for adhered platelets, collagen supported soluble thrombin formation as much as 5 times that of the thrombin-retaining clots. Activation of platelets by their firm adhesion was accompanied by formation of microparticles, representing about one third of the total soluble PCA. Collagen-adhered platelets provide soluble thrombin and microparticles, whereas the preformed clot serves to localize and accelerate hemostasis at the injury site, with the contribution of retained thrombin and microparticles.

Abciximab inhibits procoagulant activity but not the release reaction upon collagen- or clot-adherent platelets.

In addition to inhibiting platelet (plt) aggregation abciximab, a glycoprotein (GP) IIb/IIIa antagonist, reduces coagulation in blood or platelet-rich plasma. We assessed the effects of abciximab (10 micro g mL(-1)) on adhesion-dependent procoagulant activity (PCA) of plt upon: (i) collagen to model initial adhesion; or (ii) plasma clot or fibrin to model a preformed thrombus with retained thrombin activity. In a two-stage assay gel-filtered plt (GFP) first adhered on collagen, plasma clot, or fibrin, and plt activation was traced with platelet factor 4 (PF 4) release. Second, PCA was measured on adherent plt (i) by soluble prothrombin fragments (F1 + 2); and (ii) chromogenically by adding defibrinated plasma and thromboplastin. Abciximab inhibited aggregation upon collagen-adherent plt both in the absence and presence of plasma. In contrast, without plasma abciximab enhanced plt deposition to fibrin surfaces depending on thrombin generation and fibrin polymerization. However, abciximab reduced PCA and generation of F1 + 2 on adherent plt surface-independently by 35%, whereas PF 4 release persisted. Also, a GP Ib inhibitor, mAb SZ2, attenuated PCA by 40% alone, and by 65% together with abciximab, leaving 35% of PCA unaltered. Abciximab decreased generation of new thrombin on both collagen- and clot-adherent plt. However, abciximab did not inhibit alpha-granule release, suggesting distinct pathways for PCA and release reaction. Deposition of isolated plt on clots in the presence of abciximab was dependent on thrombin and polymerizing plt-derived fibrin(ogen). Due to local consumption of natural anticoagulants adjacent to a preformed thrombus the antithrombotic effect of abciximab benefits from additional inhibition of thrombin.