Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

Abstract

The role of platelets in arterial thrombosis is well established. Recent findings have reinforced their importance in venous thrombosis as well. Which of the platelet's many functions mediate its effects in the venous setting remains unclear, and how procoagulant platelet formation regulates venous thrombosis has not been investigated. Cyclophilin D (CypD) is a mitochondrial peptidylprolylisomerase that has the key function of regulation of mitochondrial permeability transition pore (mPTP) formation. In the absence of CypD, mPTP and procoagulant platelet formation are markedly impaired in response to physiologic agonists. The functional consequence of CypD's absence in vivo has been examined in photochemical injury models in photochemical injury models of both the carotid and mesenteric arteries. In mice with platelet CypD-deficiency, in both of these high shear settings, thrombotic vasoocclusion was accelerated implicating procoagulant platelet fragility and loss of adhesiveness as a critical factor limiting thrombus growth. Whether CypD deficiency would similarly impact thrombus growth in a venous setting has not been previously examined.To investigate the role of CypD and procoagulant platelets in the low shear setting of venous thrombosis, we utilized the inferior vena cava (IVC) stenosis ligature murine model of deep vein thrombosis (DVT). This model was chosen as it utilizes mechanical narrowing to induce thrombosis in the setting of venous flow. 24 hours following IVC ligation, thrombi were significantly larger (1.83 fold) in CypD-deficient mice than wild type. (p<0.05). Similarly, in mice with platelet-specific CypD-deficiency, thrombi were 1.54 fold larger than those found in WT mice (p<0.05). To begin to investigate the mechanism by which CypD-deficiency resulted in increased thrombus size, the composition of the venous thrombi was analyzed. Western blots for markers of platelets (CD41), neutrophils (Ly6G), and fibrin (59D8) revealed that their relative levels in thrombi were unchanged, but indicated that red blood cell (spectrin α1) accumulation increased. Histologic appearance in both CypD-deficient and wild-type thrombi was similar with a consistent pattern observed. A white cap area dominated the thrombus closest to the site of ligation, while red thrombus was more common in the remaining thrombus area. To investigate whether the observed pattern is due to more rapid occlusion, ultrasound was utilized to investigate the time course of venous vasoocclusion following ligature placement. Preliminary ultrasound studies suggest that CypD-deficient mice develop vasoocclusion more rapidly, perhaps thus accounting for their larger size.Previous studies have delineated the role of CypD-dependent events in limiting platelet accretion in vitro in arterial, but not venous, flow conditions. In vitro studies utilizing whole blood from CypD-deficient mice showed increased accretion of platelets on collagen under venous flow conditions. Similarly, platelet accretion increased when human whole blood was treated with cyclosporin A, an inhibitor of CypD, and flowed over collagen at venous rates. Interestingly, washed platelets from CypD-deficient mice also showed increased accumulation on a von Willebrand factor (VWF) - coated surface under venous flow conditions, a condition in which neither collagen nor coagulation-mediated procoagulant platelet formation would be expected to occur. Thus, these studies have identified an important role for CypD-dependent platelet events in the regulation of platelet accumulation in vitro and thus slowing the early stages of thrombus growth in vivo . The results here, in venous settings, and previously in arterial settings are consistent with a limiting role for procoagulant platelet formation in thrombus growth. This regulatory feedback role should be taken into consideration prior to modulation of procoagulant platelet formation in the setting of thrombosis or inflammation. DisclosuresNo relevant conflicts of interest to declare.