Thrombus Formation At Pathological Shear Rates And The Role Of Truncated Integrin Beta 3 In Integrin Bidirectional Signaling

Cardiac and cerebrovascular thrombostic diseases are mainly due to pathological thrombus formation. The physiological thrombosis develops chiefly in arteries without stenosis, whereas the pathological thrombosis occurs mainly in atherosclerotic stenotic arteries where the narrowed vessels create greatly elevated wall shear rates. Pathological high shear rates are not only the results of various pathological factors, but also the reasons of disease deterioration. However, it is not clear about the characteristics and the influential factors of thrombosis at pathological high shear rates. Using Bio Flux 200 we observed the features and influential factors of thrombus formation at different shear rates.Within a certain time on collagen-coated matrix, the total volume, the total area coverage, and the number of thrombi per unit visual field, as well as the volume and area coverage per thrombus, generated a parabolic curve-like change with the increase of shear rates, in which the value reached top at a shear rate of 2500 s-1. However, the height of thrombi manifested a linear rise with the increase of shear rates. Physiological venous shear rates generated sporadic platelet adhesion, physiological arterial shear rates generated extensive and thin platelet aggregation elongated in the direction of flow, whereas elevated pathological shear rates generated sporadic and thick thrombi, which were easy to shed to form emboli, and this shedding was relative to the level of shear rates and the heigh of thrombi. At pathological high shear rates thrombus formation needed to firstly “catch”(reversible adhesion) platelets under flow mediated by GPIb-v WF-collagen interaction, then “catch firmly”(irreversible adhesion) and activate platelets mediated by αIIbβ3-v WF interaction, lastly aggregate platelets mediated by αIIbβ3-fibrinogen interaction. The cell-permeable peptide myr-AC~CRGT which selectively impaired αIIbβ3 outside-in signaling partially inhibited thrombus formation at shear rates of 5000s-1 and 1500s-1, but did not affect the adhesion and aggregation of platelets at shear rates of 500s-1 and 125s-1. Platelet deposition and platelet activation induced by shear rates, adhesion receptors of platelet, and washing of thrombi by shear stresses composed a thrombosis-embolization balance system. Part ⅡAntithrombosis while with reserving basic hemostasis through selectively inhibition of outside-in signaling without affecting inside-out signaling is a novel strategy of antithrombotic therapies. It has been reported that β3 last three amino acids RGT are obligatory for outside-in signaling, but dispensable for inside-out signaling. However, the roles of the other β3 cytoplasmic residues, such as last 8 amino acid T755NITYRGT762 and distal N756ITY759 motif in particular, in the αIIbβ3 bidirectional signaling need to be verified in platelets, and moreover, whether the adhesion of platelets with selectively damaged αIIbβ3 outside-in signaling is affected under flow still remains unknown. We established transplanted transgenic mice with different truncated β3 cytoplasmic tails(β3-762, β3-Δ759, β3-Δ754, β3-ΔNITY, and vector) through transplanting β3-/-(β3 deficient) mouse bone marrow cells which were infected by highly infective retrovirus containing different truncated β3 cytoplasmic tails into lethal radiated wild-type mice and tested bidirectional signaling pathways at static and flow condition. GFP positive rates of transgenic mouse platelets ranged from 1% to 66%. Although the β3 expression levels of the transgenic mice varied, the function loss of the mutant transgenic platelets could not be attributed to the β3 expression levels. In β3-Δ759 platelets the inside-out signaling was not affected, while outside-in signaling was injured. Moreover adhesion of β3-Δ759 platelets under flow was partly impaired. In β3-Δ754 platelets the bidirectional signaling was obviously impaired and adhesion under flow was less. In β3-ΔNITY platelets the inside-out signaling was affected, while outside-in signaling was reserved partly with slightly decreased adhesion under flow. Conclusively, this study provided proof in platelets at static or flow condition that integrin β3 cytoplamic distal NITY motif was crucial for inside-out signaling pathway and integrin β3 C terminal last three amino acids RGT were the most important sequences attending outside-in signaling which could be developed into a potential antithrombotic target.