The essential event in platelet adhesion to the injured blood vessel wall is the binding to subendothelial collagen of plasma von Willebrand factor (VWF), a protein that interacts transiently with platelet glycoprotein Ib␣ (GPIb␣), slowing circulating platelets to facilitate firm adhesion through collagen receptors, including integrin ␣21 and GpVI. To locate the site in collagen that binds VWF, we IntroductionThe interaction of collagen with von Willebrand factor (VWF) requires unique structural properties in both proteins. Optimal hemostatic function requires multimerization of up to 50 VWF monomers in circulating plasma; higher-order multimers bind collagen more tightly than smaller assemblies of VWF. 1 Several collagens occur in the vessel wall, of which collagens I and III are considered most important in supporting platelet adhesion to the damaged vasculature. 2 We have identified the residues in the VWF A3 domain that bind collagen III, using site-directed mutagenesis guided by the crystal structure of the VWF A3 domain in complex with a monoclonal antibody (RU5) that inhibits its interaction with collagen. 3,4 Nishida et al mapped the collagen-binding mode of the A3 domain by nuclear magnetic resonance and confirmed results by site-directed mutagenesis. 5 However, the VWF-binding site(s) in collagen is unknown, although progress in understanding how collagen interacts with integrin ␣21 and GpVI has been made using short synthetic triple-helical peptide analogues of collagen, 6,7 including the Collagen III Toolkit. 8 We used the same approach to identify the high-affinity VWF-binding site in human collagen III, information that may help to develop the collagen-VWF interaction as an antithrombotic target. 9,10 Materials and methods Peptide synthesisThe synthesis and characterization of the 57 overlapping triple-helical peptides of the Collagen III Toolkit (Table S1, available at the Blood website; see the Supplemental Materials link at the top of the online article) is detailed elsewhere. 8 The same approach was used to synthesize and verify derivative peptides (Table S2). The sequence of peptide no. 23 is GPC-(GPP) 5 -GPOGPSGPRGQOGVMGFOGP-KGNDGAO-(GPP) 5 -GPC-NH 2 , and of the minimal VWF-binding derivative peptide, GPC-(GPP) 5 -GPRGQOGVMGFO-(GPP) 5 -GPC-NH 2 . Static platelet-binding assayBlood was obtained from the antecubital vein of informed volunteers, in accordance with the Helsinki protocol, into 0.105-M citrate Vacutainers (Becton Dickinson, Oxford, United Kingdom). Platelet-rich plasma was prepared after 2 spins for 1 minute at 1200g. Then, 10% (vol/vol) of ACD buffer (39 mM citric acid, 75 mM trisodium citrate, 135 mM D-glucose, pH 4.5) and prostaglandin E 1 (280-nM final concentration) were added, and the platelets were pelleted for 12 minutes at 700g, then resuspended in 6 mL buffer (5.5 mM D-glucose, 128 mM NaCl, 4.26 mM Na 2 HPO 4 , 7.46 mM NaH 2 PO 4 , 4.77 mM trisodium citrate, 2.35 mM citric acid, 0.35% bovine serum albumin [BSA], pH 6.5). Prostaglandin E 1 was added similarly, and the ...
Key Points VWFpp discriminates between type 3 VWD patients and severe type 1 VWD patients with very low VWF levels. The pathophysiological mechanisms of all types of VWD can be defined by the combined ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag.
K E Y P O I N T Sl Liver-associated tissue factor drives rapid intrahepatic coagulation after PHx.l Intrahepatic fibrin(ogen) deposition, but not thrombin-mediated platelet activation, promotes liver regeneration after PHx.Platelets play a pivotal role in stimulating liver regeneration after partial hepatectomy in rodents and humans. Liver regeneration in rodents is delayed when platelets are inhibited. However, the exact mechanisms whereby platelets accumulate and promote liver regeneration remain uncertain. Thrombin-dependent intrahepatic fibrin(ogen) deposition was recently reported after partial hepatectomy (PHx) in mice, but the role of fibrin(ogen) deposits in liver regeneration has not been investigated. We tested the hypothesis that fibrin(ogen) contributes to liver regeneration by promoting intrahepatic platelet accumulation and identified the trigger of rapid intrahepatic coagulation after PHx. PHx in wildtype mice triggered rapid intrahepatic coagulation, evidenced by intrahepatic fibrin(ogen) deposition. Intrahepatic fibrin(ogen) deposition was abolished in mice with liver-specific tissue factor deficiency, pinpointing the trigger of coagulation after PHx. Direct thrombin activation of platelets through protease-activated receptor-4 did not contribute to hepatocyte proliferation after PHx, indicating that thrombin contributes to liver regeneration primarily by driving intrahepatic fibrin(ogen) deposition. Fibrinogen depletion with ancrod reduced both intrahepatic platelet accumulation and hepatocyte proliferation after PHx, indicating that fibrin(ogen) contributes to liver regeneration after PHx by promoting intrahepatic platelet accumulation. Consistent with the protective function of fibrin(ogen) in mice, low postoperative plasma fibrinogen levels were associated with liver dysfunction and mortality in patients undergoing liver resection. Moreover, increased intrahepatic fibrin(ogen) deposition was evident in livers of patients after liver resection but was remarkably absent in patients displaying hepatic dysfunction postresection. The results suggest a novel mechanism whereby coagulation-dependent intrahepatic fibrin(ogen) deposition drives platelet accumulation and liver regeneration after PHx. (Blood. 2019;133(11):1245-1256
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