Platelet Reactivity and CD39 Ectonucleotidase Activities in Cryptogenic Stroke
Authors: Marcus Aaron J. , Broekman Marinus Johan , Drosopoulos Joan HF , Olson Kim E. , Pulte Dianne , Kizer Jorge R. 
Published: October 2019

Abstract
In the US, 780,000 people are diagnosed with stroke annually, of which 180,000 are recurrent. Nearly 90% of strokes are ischemic and 10% hemorrhagic. Roughly 30% of ischemic strokes are of unknown etiology or “cryptogenic”; this figure is even higher in younger adults. We sought to determine whether platelet activation and recruitment are increased in younger patients with cryptogenic stroke. In addition, we postulated a prothrombotic change in their endogenous CD39/NTPDase1 expression and nucleotidase activities (metabolism of ATP and ADP to AMP). Our sample consists of patients with cryptogenic stroke (n=40) and healthy controls (n=35), ages 18 to 64, participating in the THrombophilia In Cryptogenic stroKe (THICK) study. Extensive preliminary testing for humoral prothrombotic disorders did not account for almost half of the cerebral infarcts. This suggests that cryptogenic stroke may in part represent a disorder of enhanced platelet reactivity. We did indeed find that markers of platelet activation were higher in cases than controls as determined by platelet aggregation in platelet-rich plasma (lumiaggregometry) and FACS analyses. ATP secretion, a measure of platelet recruitment, was higher in stroke vs. control (no ASA) with 5 and 0.5 μg/ml collagen, and with 5 and 0.5 μM epinephrine. FACS analyses revealed that CD154, CD63, and monocyte-platelet aggregates were increased (P=0.18, 0.048 and 0.034). By contrast, neither platelet aggregation (expressed as area under the curve) in response to 0.5 μg/ml collagen nor circulating tissue factor activity were significantly higher in cases as compared to controls. Together these findings suggest a potential role for enhanced platelet activation and recruitment in younger cryptogenic stroke patients. In addition, we established that CD39/NTPDase1 is expressed on neutrophils (PMN), lymphocytes, and monocytes with ATPase and ADPase activities highest on B-lymphocytes, lower on PMN, lowest on T-lymphocytes. In stroke subjects, trends to higher total ADPase activities were observed in lymphocytes (P=0.09) and PMN (P=0.09). In contrast, ATPase activities were similar (P=0.81 and 0.68). Thus, the ratio of ADPase to ATPase activity was greater in stroke patients than controls (P=0.003 for lymphocytes; 0.13 for PMN) in apparent compensation for prothrombotic propensities. In general, these same trends were observed for direct comparisons of stroke patients to controls, whether on ASA or not. This is in apparent contrast to the data obtained previously with CAD patients in the acute phase (El-Omar et al, Thrombosis Res.116:199–206, 2005), and supports the need for additional studies of cryptogenic and atherothrombotic stroke patients in both acute and convalescent phases. Total CD39 expression (FACS, assessed with mAb BU61) was only marginally higher in lymphocytes of patients as compared to controls. The finding of increased activities, but similar expression supports our hypothesis that the enzymatic nucleotidase activities of CD39 are regulated via expression of our newly discovered CD39 splice variants. Ultimately, therapy with solCD39 to block platelet activation and recruitment may hold promise for patients with cryptogenic stroke.