Tetraspanin CD37 Regulates Platelet Hyperreactivity and Thrombosis

Psychosocial stress increases the risk of a myocardial infarction 2-fold and is associated with a 40% higher risk of death following an acute thrombotic event. With approximately a third of US adults reporting overwhelming stress levels most days, defining the mechanisms by which stress contributes to adverse thrombosis is paramount. Platelets are key contributors to thrombotic cardiovascular events, with platelet hyperreactivity associated with thrombosis and psychosocial stress. In humans and mouse models, psychosocial stress induces a hyperreactive platelet phenotype and increases the risk of thrombosis.

To investigate how stress contributes to platelet hyperreactivity, platelets were isolated from 1) mice that experienced chronic variable stress and stress-free controls (n=8/group) and 2) human subjects with self-reported high and no stress levels (n=18/group) and RNA-sequencing performed. By comparison of mutually expressed transcripts, we identified a subset of genes differentially expressed following psychosocial stress in both human and mouse platelets. To assess the contribution of the identified transcripts to platelet functional responses that underlie thrombosis, the association of identified genes with platelet aggregation to epinephrine (0.4 μM) was assessed. Amongst screened candidates, the tetraspanin CD37 was significantly associated with platelet aggregation in a cohort of participants whose platelet aggregation and platelet transcriptome were measured simultaneously (R=0.30, p=0.0002, n=145).

Following confirmation of CD37 expression in platelets at the transcriptomic and protein level in humans and mice, we assessed the functional outcome of platelet CD37 deficiency. Cd37^–/– platelets spread significantly less relative to wild-type platelets on both poly-D-lysine and fibrinogen (p<0.01 and p<0.005, respectively) and underwent a minimal change in morphology following fibrinogen adhesion. Platelet solidity, a proxy measure inversely proportional to efficient maximal spreading mediated by filopodial-to-lamellipodial transitions, was significantly decreased in wild-type platelets relative to Cd37^–/– (p<0.0001), indicative of a spreading dysfunction in CD37 deficient platelets.

In whole blood flood cytometry, platelet expression of activated integrin αIIbβ3 (JON/A) and CD63 is significantly attenuated on Cd37^–/– platelets relative to wild-type following stimulation with PAR4-AP (p<0.0001, p<0.05, respectively). However, JON/A and CD63 expression is unaltered under resting conditions between Cd37^–/– and wild-type platelets. Consistent with a role for CD37 in regulating thrombosis-relevant platelet activation responses, chimeric mice that received Cd37^–/– bone marrow experienced a significantly increased time to vessel occlusion (p<0.05) in the carotid artery FeCl₃ model, relative to mice reconstituted with wild-type bone marrow. CD37 deficiency does not alter hemostasis, as platelet count, coagulation metrics, prothrombin time, and partial thromboplastin time do not differ in Cd37^–/– mice relative to wild-type mice. Consistent with this, bleeding time does not differ between wild-type and Cd37^–/– mice following tail tip transection.

In humans and mice, platelet CD37 positively associates with platelet aggregation and phenotypic responses that underlie thrombosis. Cd37-deficient platelets exhibit impaired integrin αIIbβ3 signaling, characterized by reduced platelet fibrinogen spreading and decreased agonist-induced αIIbβ3 activation. Notably, CD37 deficiency significantly reduces thrombotic risk without altering hemostasis or bleeding susceptibility. This study provides new insight into a platelet-associated stress-induced mechanism contributing to accelerated thrombotic risk and provides the first direct evidence for CD37 as a regulator of platelet activation responses and thrombosis.