Summary The natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.
S ummary . To obtain information on the immunological relationship between the endothelial and platelet glycoprotein (GP)Ia‐IIa (VLA‐2) complex, we studied whether endothelial GPIa‐IIa was able to express the platelet GPIa‐IIa‐associated Br‐alloantigen system. Therefore, we tested antisera to both allelic forms of the Br system (Br a and Br b ) on platelets (by an assay based on monoclonal antibody‐specific immobilization of platelet antigens, MAIPA) and on cultured umbilical vein endothelial cells (by immunoprecipitation experiments) from the same individual. Endothelial cells from a platelet Br(a + b +), and from a platelet Br(a ‐ b +) individual were studied. Our results indicate that endothelial GPIa‐IIa is indistinguishable from platelet GPIa‐IIa in its ability to express the Br a and Br b alloantigens. The association of Br alloantigens with endothelial GPIa‐IIa was confirmed by the results of an assay based on monoclonal antibody‐specific immobilization of endothelial antigens (MAIEA). These data further illustrate the structural and immunologic similarity of platelet and endothelial cell GPIa‐IIa (VLA‐2).
Endothelial cells are able to support the activation of coagulation factor X by activated factor IX in the presence of its cofactor, factor VIII. We have previously reported that this reaction is persistent on endothelial cells, but transient on activated platelets and phospholipid vesicles when activated factor X (Xa) is used as activator of factor VIII. Aim of the present study was to explore the influence of von Willebrand factor and that of the factor VIII activator, either factor Xa or thrombin, on the decay of factor X activation on the endothelial cell surface. Kinetics of factor X activation on human umbilical vein endothelial cells was compared with that on phospholipid vesicles employing purified coagulation factors from plasma as well as recombinant factor VIII variants. Employing factor Xa as factor VIII activator, rate constants for decay of membrane-bound factor X activation were consistently low on endothelial cells (0.02 min) as compared with phospholipid vesicles (0.2 min). Activation of factor VIII by thrombin resulted in two-fold increased decay rates. In the presence of excess of von Willebrand factor over factor VIII, decay rates were not significantly changed. Factor VIII variants with and without a Tyr to Phe substitution, which abolishes high-affinity binding to von Willebrand factor, displayed the same factor X activation decay kinetics. Although previous studies have shown that von Willebrand factor modulates factor VIII activation and stabilisation, this apparently does not affect the progression of factor X activation at the endothelium.
Hemostasis and bleeding are difficult to measure. Thrombin generation assays (TGAs) can measure both procoagulant and anticoagulant contributions to coagulation. TGAs might prove useful for the study of bleeding disorders. There has been much progress in TGA methodology over the past two decades, but its clinical significance is uncertain. We will undertake a scoping review of the literature to synthesize available information on the application of TGAs towards the study of bleeding and hemostasis, TGA methodologies being used and to summarize available literature on associations between TGA parameters, bleeding and hemostatic outcomes.MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials (CENTRAL) will be searched in collaboration with an information specialist. Title/abstract and full-text screening will be carried out independently and in duplicate; eligible study types will include randomized controlled trials, non-randomized studies, systematic reviews, and case series reporting TGA results and bleeding/hemostatic outcomes among humans. Mapping the information identified will be carried out with results presented using qualitative data analytical techniques.This scoping review will use published, publicly available information. Research ethics approval will not be required. We will disseminate our findings using conference presentations, peer-reviewed publications, social media, and engagement with knowledge users. This review will outline knowledge gaps concerning TGAs, better delineate its applicability as a clinically relevant assay for bleeding. and seek to identify ongoing barriers to its widespread adoption in clinical research, and eventually, in the clinical setting.Registration ID with Open Science Framework: osf.io/zp4ge.
BACKGROUND Hyperfibrinolysis has been observed in patients heavily transfused with solvent/detergent‐treated pooled plasma (S/D plasma). We compared coagulation and fibrinolytic variables in blood containing S/D plasma with blood containing fresh‐frozen plasma (FFP), with and without α2‐antiplasmin or tranexamic acid (TXA) supplementation. STUDY DESIGN AND METHODS Whole blood samples were reconstituted from red blood cells, platelet (PLT) concentrates, and varying mixtures of FFP and S/D plasma. Hematocrit and PLT count of reconstituted whole blood samples were varied. For a subset of runs, α2‐antiplasmin or TXA was added to S/D plasma whole blood samples. Thromboelastography (TEG) analysis was performed to assess 50% clot lysis time (CLT 50% ), maximum amplitude (MA), and initial clotting time (R‐time). RESULTS The change in CLT 50% of whole blood as the plasma compartment transitions from FFP to S/D plasma was −52% (95% confidence interval [CI], −60% to −45%; p < 0.001). PLT count strengthened the effect, leading to an additional change in CLT 50% of −8% (95% CI, −14% to −2%; p = 0.012) as PLT count increased from 10 × 10 9 to 150 × 10 9 /L. MA and R‐time were not associated with fraction of S/D plasma in whole blood. α2‐Antiplasmin and TXA restored clot lysis time in S/D plasma whole blood. CONCLUSION Whole blood with S/D plasma has shorter clot lysis times in vitro compared to whole blood with FFP. α2‐Antiplasmin and TXA restore clot lysis time of S/D plasma whole blood to that of FFP whole blood. Clinicians should be aware of the decreased clot lysis time associated with S/D plasma transfusion.
