Inhibit the calpain to climb the mountain

Abstract

In this issue of Blood, Tyagi et al shed some light on the mechanism of thrombosis induced by a high altitude, hypoxic environment.¹ Using proteomic analysis of platelets and in vivo models of thrombosis, the authors elegantly demonstrated that enhanced calpain activity, regulated by CAPNS1, significantly contributes to platelet reactivity and thrombosis under hypoxic conditions. The observations from animal models were further supported by human data showing increased calpain activity and elevation in markers of platelet activation in the plasma of patients who developed deep vein thrombosis at high altitude.

Who does not know about the increased risk of developing deep vein thrombosis during long air travels? Or heard about pulmonary or cerebral thrombosis causing death of people trekking Himalaya? Have you ever thought that calpains may have something to do with that? Calpains are calcium-dependent, nonlysosomal cysteine proteases expressed ubiquitously in mammals. There are 2 major forms of calpains, the μ-calpain (also called calpain-1) and the m-calpain (calpain-2), which require micro- or millimolar calcium concentration for full activation, respectively. The calpain proteolytic system includes the calpain proteases, the small regulatory subunit CAPNS1, and the endogenous calpain-specific inhibitor, calpastatin. A previous study with recombinant calpastatin provided the first demonstration of the role of calpain in platelet secretion, aggregation, and spreading.² Subsequently, another group used μ-calpain–knockout mice to demonstrate attenuated thrombin- and adenosine 5′-diphosphate–induced platelet aggregation and clot retraction.³ Because current calpain inhibitors are unable to differentiate between μ- and m-calpain, it is unknown which calpain plays the dominant role in hypoxia-mediated platelet activation and thrombosis. Data from a recent study examining the effect of calpain on the platelet proteome and reactivity in diabetes mellitus suggest that m-calpain may be primarily involved in platelet spreading, whereas μ-calpain primarily contributes to platelet adhesion.⁴

Platelet activation and aggregation are critical for clot formation, and attenuation of these functions certainly contributed to reduction of thrombus size in hypoxic rats treated with calpain inhibitors.¹ However, additional mechanisms could also influence the outcome of this study. Recently, much attention was focused on procoagulant microparticles released from many cell types, including platelets, and their contribution to activation of coagulation and thrombosis. Interestingly, the calpain-dependent release of procoagulant microparticles from platelets has been previously described.⁵ In addition, tissue factor (TF) expressed by monocytes has been shown to promote thrombosis in a mouse model of oxygen deprivation.⁶ Of note, calpain inhibition can attenuate the activation of the nuclear factor κB pathway that plays a critical role in the regulation of TF gene expression.⁷ Unfortunately, the authors did not analyze monocyte TF expression in their study.

Another interesting aspect of the paper by Tyagi and colleagues is the demonstration of TF in rat platelets.¹ The authors observed the splicing of TF pre–messenger RNA (pre-mRNA) and increased levels of TF protein in platelets isolated from hypoxic rats. The splicing of TF pre-mRNA was previously shown in activated human platelets⁸; however, the ability of platelets to synthesize TF protein is still debated. For example, it has been proposed that the presence of TF on platelets isolated from patients with sepsis and acute coronary syndromes most likely results from the docking of monocyte-derived TF-bearing microparticles rather than de novo synthesis.⁹ Because hypoxia upregulates TF expression on monocytes,⁶ the same mechanism could contribute to the increased levels of TF protein in platelets isolated from hypoxic rats.

This study raises a number of interesting questions regarding the role of calpain and platelets in hypoxia-induced thrombosis. What is the relative contribution of the 2 major forms of calpain to hypoxia-induced thrombosis? The tail-bleeding time was not affected in μ-calpain–knockout mice.³ Would that suggest that specific targeting of this calpain can effectively reduce thrombosis without interfering with hemostasis? In addition, follow-up studies should provide more mechanistic insight into how calpains regulate platelet responses during hypoxic conditions. Finally, in contrast to rat platelets, pre-mRNA for TF was not observed in mouse platelets, precluding the use of mouse models to study the role of platelet TF.¹⁰ Because genetically engineered rat strains are now being routinely produced, it would be of interest to generate rats with conditional, platelet-specific deletion of the TF gene to address the controversy surrounding the role of platelet TF in thrombosis.