Thrombin Generation by Apoptotic Vascular Smooth Muscle Cells

Abstract

Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 ± 17 nmol⋅min/L and a peak thrombin (PT) of 154 ± 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 ± 15 nmol⋅min/L, P = .3; PT of 172 ± 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 ± 8 nmol⋅min/L; PT of 103 ± 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 ± 2 nmol⋅min/L; PT of 128 ± 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.

Thrombogenic role of cells undergoing apoptosis

“…The protocol was modified from the method reported by Flynn et al (1997). Briefly, blood from healthy individuals was used to prepare platelet-poor plasma by centrifugation at 3000 g for 15 min.…”

Abnormalities in hemostasis in acute promyelocytic leukemia

“…We have observed that NB4 cells in culture generate larger amounts of thrombin than any other leukemic cell line. 16 Using the method of Flynn et al, 17 cell suspensions were mixed with defibrinated plasma, Russell's viper venom, and the coagulation process was activated with calcium chloride. The thrombin generated was determined by chromogenic substrate assay.…”

Vascular smooth muscle cell apoptosis in atherosclerosis

“…Apoptosis of monocytes releases large amounts of the cytokine IL-1b (Miwa et al 1998); apoptotic cells are phagocytosed by binding to receptors such as CD14 (lipopolysaccharide receptor) ligation of which activates phagocytes (Devitt et al 1998). Additionally, apoptotic cells expose the inner lea¯et phospholipid phosphatidylserine externally, conferring procoagulant activity (Flynn et al 1997).…”

“…Firm evidence for either of these proposals is not vet available. Apoptotic VSMCs and endothelial cells may also promote thrombosis directly, by exposure of phosphatidylserine on their surface which may promote the formation of prothrombinase and therefore thrombin (Flynn et al 1997). The potency of this property in vivo is unknown but the ability to generate thrombin is likely to be a universal property of apoptotic cells.…”