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Fibrin forms the cohesive network of hemostatic plugs and thrombi, and it also provides the temporary matrix for initial support of healing and revascularization. Because cell proliferation is needed for revascularization after vessel injury, we have characterized structural requirements of fibrin needed to support cell proliferation on fibrin in vitro. Proliferation of cultured human endothelial cells and fibroblasts was measured by 3H-thymidine incorporation on fibrin surfaces varying in structure. Fibrin prepared with thrombin and lacking both fibrinopeptides A and B (desAB fibrin) supported proliferation of both endothelial cells and fibroblasts. In contrast, fibrin prepared with reptilase, which cleaves only fibrinopeptide A, supported significantly less proliferation. Also, fibrin prepared by thrombin treatment of fibrinogen lacking residues beta 1-42 supported only a low level of proliferation. Therefore, fibrinopeptide B cleavage and exposure of beta 15-42 enhanced proliferation of cells on fibrin. Specific proteolytic inhibitors were used to eliminate the potential mitogenic effects of residual fibrin-bound thrombin. Additional controls showed that neither catalytically inactive thrombin nor addition of the thrombin receptor-activating peptide (SFLLRNPNDKYEPF [SFLL]) stimulated proliferation on desA fibrin. The results indicate that cell proliferation on fibrin is enhanced by fibrinopeptide B cleavage and exposure of the amino terminus of the fibrin beta chain. They also show that specific structural features of the temporary fibrin matrix formed at sites of injury may modulate the proliferative response of vascular cells.

Endothelial cells (EC) interact with fibrin at sites of vascular injury, thrombosis, inflammation and tumor growth, whereas they are quiescent when exposed to circulating fibrinogen. To determine the structural basis for specific interaction with fibrin we have characterized the response of EC to fibrin of varying structure. Fibrin was prepared with thrombin, which cleaves both fibrinopeptide A (FPA) and fibrinopeptide B (FPB), with Reptilase, which cleaves only FPA, and with contortrix procoagulant to cleave only FPB. Fibrin with FPB cleavage stimulated release of von Willebrand factor from EC Weibel-Palade bodies and also supported cell spreading. Involvement of the amino terminus of the fibrin beta chain in the response was shown by stimulation of von Willebrand factor release by the peptide beta 15-42. Also, fibrin prepared from a fibrinogen derivative lacking residues 15-42 of the beta chain failed to support EC spreading. EC adhesion was unaffected by the pattern of fibrinopeptide cleavage or by the removal of peptide beta 15-42 from fibrin. The results indicate that separate sites on the fibrin molecule mediate adhesion and spreading, and that the latter requires cleavage of FPB and the new amino terminus of the beta chain. They further suggest that cellular responses to fibrin are regulated by the proteolytic cleavages and conformational changes that convert fibrinogen to fibrin and may also be modulated by plasmic or elastase degradation.

Adhesion and spreading of cultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin beta chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B beta chain. After clotting with thrombin, this fibrin derivative lacking B beta 1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 15-42 of the fibrin beta chain.