Biomechanical Platelet Activation: Diseases that Require a New Class of Antiplatelet Therapeutics

Abstract

Mechanisms of platelet activation have traditionally been investigated through the activation of biochemical pathways through cell surface agonists such as ADP, thrombin, and collagen. However, recent research has identified another crucial mechanism—biomechanical activation, where external physical forces directly influence platelet reactivity. This paradigm shift underscores the complex interplay between biochemical and biomechanical stimuli in platelet activation. This review aims to understand the molecular mechanisms underlying biomechanical activation and the implications for treating thrombotic disorders.

Introduction

Platelet activation plays a pivotal role in balancing the protective physiological mechanism of hemostasis whilst limiting the thrombosis which is a pathological event. Although anucleate cytoplasmic fragments, platelets possess extracellular receptors and adhesion molecules and can synthesize proteins that permit them to sense their environment, interact directly, and respond effectively. (1,2) Most molecules released from platelets during activation are preformed in bone marrow megakaryocytes, highlighting their pre-programmed role in thrombosis. (1)

Platelet activation is primarily coordinated with activation of the coagulation cascade when responding to vascular injury involving interactions with the exposed subendothelium, adhesion to collagen with the platelet glycoprotein VI (GPVI) receptor and αIIβ1 integrin, and ligation of the platelet receptor GPIbα complex with Willebrand factor (vWF). Platelets accumulate and aggregate with each other via surface integrin αIIbβ3 (also known as the GPIIb/IIIa), promoting luminal thrombus formation in vivo. This pathological process is further amplified by Factor IIa (thrombin)-mediated platelet activation through platelet surface Protease-Activated Receptor 1 (PAR1) and PAR4. (3,4)

Thrombosis can also occur independently of platelet membrane receptor agonists in undamaged blood vessels, often in areas with high shear stress due to stenosis. Mechanotransduction, or mechanosensitive activation, is a process whereby platelets respond to external mechanical forces, transducing physical stimuli into downstream biochemical signals inside the platelet that amplify platelet activation. Recent advancements in biophysical technologies, including molecular imaging and live-cell dynamic force spectroscopy, have uncovered emerging pathways of platelet adhesion, spreading, and aggregation mediated by shear stress. (5,6) These techniques have revealed how platelets, similar to nucleated cells, interact dynamically with their mechanical microenvironments, such as changes in vessel geometry due to atherosclerotic lesions. When there is a discontinuity in the intima of blood vessels, physiological steady laminar flow (S-flow) becomes pathologically disturbed flow (D-flow). D-flow serves as an external stimulus of mechanotransduction, influencing discoid platelet aggregation and thrombus formation. (7,8)

Several in vitro studies simulating the in vivo microenvironment revealed that mechanical stress activates platelet receptors through glycoprotein and integrin interactions. (9) Identified platelet mechanoreceptors include the VWF receptor GPIb-IX complex which exists in a 1:1 stiochiometric ratio, fibrin(ogen) receptor GPIIb/IIIa GPVI, and αIIβ1 integrin. Shear stress may also activate the mechanosensitive platelet calcium channel Piezo 1. Piezo 1 promotes rearrangement of the sub-membrane actin cytoskeleton directly and in coordination with αIIbβ3 integrin, suggesting that physiological cross-talk exists. (9,10) Additional mechanosensitive channels that are indirectly activated by the Piezo 1 channel include P2X (purigenic channel X1), TRPC6 (transient receptor potential C6), or ORAI1 (calcium release-activated calcium modulator 1). (10,11,12,13)

Despite advancements in cardiovascular care, thrombotic diseases, including acute coronary syndromes, ischemic stroke and peripheral arterial disease, remain a leading cause of death and disability worldwide, where platelets play a central role in instigating arterial thrombosis. Additionally, it has been recognized that arterial thrombosis is regulated by biomechanical factors, particularly pathological shear stress and flow disturbance associated with vessel stenosis induced by atherosclerotic plaques or implanted medical devices as part of mechanical circulatory support in patients with heart failure. (14,15) Furthermore, non-atherosclerotic diseases such as fibromuscular dysplasia (FMD) may exhibit platelet hyperreactivity as a consequence of external platelet mechanical stress given the prevalence of D-flow circulation in FMD. Biomechanical platelet activation may not be effectively managed by traditional antiplatelet drugs like aspirin. (16) Newer studies have also suggested that platelet hyperactivity is implicated in neurodegenerative diseases such as Alzheimer’s Disease and Parkinson’s Disease. (17,18)

At this time, contemporary FDA-approved antiplatelet agents target only biochemical agonist pathways for platelet activation, including the purinergic P2Y₁₂ receptor antagonists (clopidogrel, prasugrel, ticagrelor), inhibitors of thromboxane A₂ (TxA₂) generation (aspirin, triflusal) or the protease-activated receptor 1 (PAR1) antagonist vorapaxar. These approved antiplatelet therapies, while effective, increase bleeding risk and may not adequately address biomechanical platelet activation. This compels us to develop novel therapeutics to decrease residual thrombotic risk. (19)

Main-Section

Platelet Activation through Mechanotransduction

In healthy conditions, platelets adapt to various mechanical forces but become prothromboticregions of the vasculature that are diseased (atherosclerotic stenosis/non-atherosclerotic, inflammatory stenosis) due to an increase in shear stress generated at the region of bifurcation in blood vessels physiologically or by atherosclerosis pathologically. Biomechanical platelet activation can be quantified by increased expression of platelet surface markers such as CD62P (P-selectin) along with conformational changes in glycoprotein and integrin levels, which are not observed during S-flow conditions. Platelet mechanoreceptors transduce mechanical stress signals and rearrange the sub-membrane actin cytoskeleton, enhancing calcium influx, which further promotes platelet degranulation and activation. (20, 21)

In addition to shear stress, platelets can sense the stiffness of their external environment by interacting with extracellular matrix (ECM) proteins. (22) Increased stiffness of the ECM promotes platelet activation, as measured by conformation changes in GPIIb/IIIa, α-granule exocytosis (surface CD62P exposure), and procoagulant platelet activity (surface Annexin V exposure). This substrate stiffness-dependent platelet adhesion is mediated by Rac1, actin, and myosin activity. (23) Platelet mechanical forces sensed by different mechanoreceptors lead to a change in platelet shape from rearrangements of filamentous actin and increased adhesion to the ECM.

Platelet Mechanoreceptors:

Piezo1—

Piezo1, a mechanosensitive ion channel located in the plasma membrane of some cells, is an important mediator of mechanotransduction. (24,25,26) Piezo1 is expressed in a variety of cell types and tissues, including endothelial cells, platelets, and platelet precursor megakaryocytes (MKs). (24,27,28). Piezo1 is activated by shear stress and the Piezo1 agonist, Yoda1, permitting calcium entry and activation of calcium-dependent signaling pathways. (21,27)

Piezo1 can sensitize other platelet mechanosensors, including the αIIbβ3 integrin receptor. (29) Piezo1 has been shown to activate calpain, a cytosolic Ca-^2+-activated cysteine protease. (30,31) This, in turn, leads to talin activation and increased inside-out signaling of the αIIbβ3 receptor. (32) Additionally, Piezo1 inhibition with XueShuanTong (XST) attenuates calpain activation and talin cleavage. Furthermore, Piezo1 activation with Yoda1 and inhibition with GsMTx‐4 were shown to influence αIIbβ3 activation and platelet surface CD62P expression. Piezo1 may also regulate the PI3K/AKT signal transduction pathway. Zhao et al. showed that piezo1 inhibition reduced the expression of phosphatidylinositol 3-Kinases (PI3K) and protein kinase B (AKT). (29) The PI3K/AKT pathway has been implicated in platelet activation and thrombosis. (33) An elegant study by Evtugina et al. showed that piezo1 activation increased thrombin formation and led to enhanced clot contraction. (34)

Piezo1 overexpression and activation have been described in diseases that constitute cardiovascular risk. In patients with diabetes, piezo1 activity was increased in platelets, red blood cells and neutrophils. Hyperglycemia was found to increase gene expression of piezo1 in cells, and inhibition of piezo1 was protective against thrombosis in human blood and zebra genetic fish models. (35) Piezo1 expression was also found to be increased in hypertensive mice, where, curiously, the authors coincidentally reported increased platelet aggregation. Furthermore, piezo1 inhibition led to a decrease in platelet adhesion and suppressed carotid thrombosis in hypertensive mice. (29)

GPIbα Receptor—

vWF is a large glycoprotein synthesized in MKs and stored in α-granules, serving as a natural ligand for activation of GPIbα, promoting platelet-to-platelet adhesion and thrombus formation. GPIbα is also synthesized in endothelial cells and released upon endothelial cell stimulation. GPIbα consists of 275kDa disulfide-linked subunits, with each subunit containing domains (primarily A1, A2, A3, and C1) mediating interactions with platelets, Extracellular matrix and plasma proteins. Newly formed vWF, when released into circulation, primarily exists in large multimers, which are highly prothrombotic. ADAMTS13 cleaves these ultra-large vWF multimers to a less adhesive form as it adopts a globular conformation. (36,37)

On the other hand, glycoprotein GPIbα is a part of platelet integrin complex GPIb-IX-V, which consists of 3 other transmembrane proteins, including GPIbβ, GPIX, and GPV. (38)

Under low shear stress conditions, vWF is auto-inhibited and cannot bind to mechanoreceptors on platelets. In high shear stress, vWF unfolds to switch to an extended conformation, leading to the interaction of transmembrane glycoprotein GPIbα on platelets with the A1 domain. Additionally, the A3 domain binds to Col 1 (collagen 1), which in turn leads to the interaction of integrin GPIIb/IIIa and the C1 domain. In conditions with very high shear rates (>10,000.sec⁻¹), such as arterial stenosis, GPIbα-A1 interaction plays a critical role and serves as the primary mediator in platelet activation. Further, the hydrodynamic forces strengthen this bond, causing platelet rolling, slowing down and arresting the vessel wall. Using single-cell force spectroscopies, researchers explained this phenomenon through the concept of ‘catch bond’ behavior. (39,40)

Furthermore, the binding of vWF to GPIbα triggers intracellular signaling, leading to intraplatelet calcium flux and subsequent activation of GPIIb/IIIa integrin. Inhibition of the GPIbα-triggered Ca²⁺ prevents platelet adhesion, suggesting that Ca²⁺ is an obligatory second messenger in the GPIIb/IIIa activation pathway. Intracellularly, a scaffold protein that binds to GPIbβ cytoplasmic tail, 14-3-3ζ, plays a critical role in downstream signaling pathways upon vWF binding, leading to the activation of different kinases, phosphatases, and adaptors primarily including Src (spleen tyrosine kinase), family kinase Lyn, and PI3K (phosphoinositide 3-kinase). (39,40,41)

Other adaptor molecules, such as FLNA (filamin-A), further cross-link the actin cytoskeleton and stabilize the interaction. (40) Murine studies showed that platelets with defective filamin activity resulted in impaired platelet adhesion at high shear. Another protein that regulates the actin cytoskeleton, COTL1 (coactosin-like protein 1), has been identified, and the shear-dependent thrombus formation was found to be altered in COTL1-deficient mouse platelets. (42). While the interaction of GPIbα-A1domain is well-understood mechanotransduction, the role of other glycoproteins such as GPIX and GPV remains unclear.

GPIIb/IIIa Integrin/Platelet-fibrin(ogen) Receptor—

αIIbβ3 integrin is another major mediator of mechanotransduction in both thrombosis and hemostasis. This transmembrane protein is abundant on the platelet membrane in its inactive form, adopting a bent conformation with a closed headpiece. The αIIbβ3 integrin serves as a connection between fibrin(ogen) and platelet actin cytoskeleton. Upon activation, ectodomain expands to extended-open (EO) conformation to achieve an extended-closed (EC) conformation. (43) The intracellular PI3K signaling pathway, which regulates vWF A1-GPIbα activation, also results in the EC conformation and, thus, intermediate affinity activation of GPIIb/IIIa integrin. Once activated, GPIIb/IIIa integrin mediates bidirectional signaling, leading to self-activation to a high-affinity state, EO conformation. This further leads to enhanced platelet spreading, calcium influx, and granule secretion. This mechanical activation of GPIIb/IIIa was observed without a soluble agonist. Only the intermediate-affinity state can undergo outside-in mechanosignaling, thus serving as auto-inhibition to prevent excessive platelet activation in the presence of normal mechanical shear force. (43)

Furthermore, soluble fibrinogen binds to GPIIb/IIIa and becomes immobile on the platelet surface. Thrombin converts fibrinogen to fibrin, exposing the cryptic GPIIb/IIIa binding site and promoting further fibrin mesh formation and mechanical stiffness. The more the stiffness of fibrin(ogen), the more the platelet adhesion and spreading. This platelet mechanosenser senses substrate stiffness in the ECM and leads to further downstream signaling involving Rac1 (Rac family small GTPase 1). (44,45) Mechanoredox coupling has also been identified as an activation mechanism of GPIIb/IIIa. Inside activated platelets, redox-sensitive enzymes such as thiol isomerases (protein disulfide isomerase/ PDI, endoplasmic reticulum 5/ERp5 and ERp57) are secreted to the surface, leading to disruption of disulphide bond on the β3-tail of GPIIb/IIIa coupled with a talin-linked protein complex. (45) This complex interaction regulates the movement of actomyosin-mediated internal mechanical force, leading to enhanced platelet aggregation and spreading.

Glycoprotein VI Receptor—

The GPVI receptor is expressed only on platelets, which was first described by Nieswandt et al. (47). In addition to being activated by collagen, GPVI also serves as a mechanoreceptor in platelets and precursor MKs. GPVI is activated by extracellular collagen in the subendothelial extracellular matrix in response to tissue injury as well as exogenous collagen-related peptide (CRP), which in turn causes phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the associated FcR gamma-chain by Src family kinases (SFKs). In addition to binding to collagen, GPVI triggers GPIIβ/IIIα and integrin α2β1 interaction, resulting in platelet aggregation. (48,49). Furthermore, animal studies suggested that GPVI-deficient mice showed impaired thrombus formation under high-shear conditions. Intriguingly, vWF-GPIbα-mediated platelet interaction was also impaired in GPVI-deficient mice, possibly suggesting co-association between GPVI and GPIbα. (50) Further research is crucial to unravel the mechanism behind this cross-talk. A summary of platelet mechanosensors and associated diseases in the primary literature is summarized in Table 1 and platelet mechanorecepotor function is indicated in the Graphical Abstract.

Table 1:

Summary of various platelet mechanoreceptors/ mechanosensor/ channels and disease association

Platelet Mechanosensor	Key Features	Group/Year	Disease Association	Citation
Piezo 1	Mechanosensitive ion channel mediating shear-dependent Ca²⁺ influx and thrombus formation in human platelets	Ilkan et al/ 2017	Increased thrombosis risk, especially in conditions like hypertension and diabetes	29
GPIbα	Acts as primary force sensor during vWF binding; LRRD prolongs bond lifetime under force, while MSD triggers intracellular Ca²⁺ signaling via unfolding. Critical for shear-dependent platelet adhesion.	Ju, Chen et al./ 2016	Increased thrombosis risk especially ischemic cerebrovascular disease, pulmonary thromboembolism; Bernard-Soulier Syndrome	42
αIIbβ3 Integrin	Functions as anisotropic mechanosensor; synchronizing calcium flux and adhesion stabilization under shear.	Zhang et al./ 2016	Glanzmann thrombasthenia (absence of platelet aggregation), thrombosis	46
GPVI	Collagen-induced platelet activation and aggregation; initiates platelet adhesion to exposed collagen after vessel wall injury	Nieswandt and Watson in 2003	Elevated levels associated with various thrombotic and inflammatory conditions (e.g., acute ischemic stroke, atrial fibrillation, sepsis)	47

Figure 1 and Graphical Abstract Legend:

This figure illustrates key mechanotransduction pathways that regulate platelet activation under shear stress.(1) Shear stress induces a conformational change in vWF, exposing its A1 domain. (2) The activated vWF binds to the GPIb-IX-V receptor complex, initiating platelet adhesion. (3) The adaptor protein 14-3-3ζ plays a key role in transducing intracellular signals. (4) Downstream activation of Src kinase, Lyn kinase, and PI3K leads to a rise in intracellular Ca²⁺ levels. (5) Increased Ca²⁺ triggers platelet degranulation, which releases numerous factors, including pro-coagulant factors. (6) Cytoskeletal proteins such as COTL1 and Filamin A (FLNA) cross-link the cytoskeleton, stabilizing platelet adhesion. (7) Calpain and talin activation further strengthen cytoskeletal remodeling and integrin signaling. (8) The GPIIb/IIIa integrin undergoes a conformational shift from its inactive (EC) to active (EO) state, enabling fibrinogen binding. (9) Soluble fibrinogen binds to GPIIb/IIIa, facilitating platelet aggregation and thrombus formation.

Additionally, mechanosensitive ion channels contribute to platelet activation. (A) Shear stress activates the Piezo1 channel, (B) leading to Ca²⁺ influx, which (C) activates kinases and subsequent downstream proteins involved in cytoskeletal reorganization and GPIIb/IIIa integrin activation (steps 6–9). Furthermore, collagen-mediated signaling plays a critical role in platelet function. (A.1) The GPVI receptor is activated by extracellular collagen in the subendothelial matrix following vascular injury. (B.1) GPVI signaling enhances GPIIb/IIIa integrin activation, promoting platelet aggregation.

Conclusions

Recent advancements in platelet mechanobiology have revealed pathways to target arterial thrombosis that are resistant to conventional antiplatelet therapies. Central to this mechanobiological process is the intricate interplay of glycoproteins and the mechanosensory domain, which transmit extracellular mechanical signals to downstream effectors regulated by complex hydrodynamics. Ongoing research targeting PI3K, Piezo1-mediated calcium influx, vWF, GPIbα, GPVI or PDI aims to decouple the cross-talk between platelet receptors and channels under high shear stress. A limitation in many of the studies in the literature that evaluate platelet mechanobiology is the reliance on highly-controlled in vitro and ex vivo experimental studies. Many aspect of platelet mechanotransduction including exposure to circulation hormanes, tightly controlled pH, temperature, viscosity of blood, and exposure of platelets to other circulating cells including eruthrocytes and leukocytes that impact platelet function are lost ex vivo. This will always be a limitation in the development of the next generation of antiplatelet drugs to mitigate biomechanical platelet activation. Mitigating these limitations would potentially offer a more targeted approach with reduced bleeding risks associated with conventional antiplatelet drugs.

Abbreviations:

ADP

Adenosine Diphosphate

GP

Glycoprotein

vWF

von Willebrand Factor

αIIβ1

Integrin Alpha 2 Beta 1

αIIbβ3

Integrin Alpha 2b Beta 3

D-flow

Disturbed Flow

S-flow

Laminar Flow

VWF

von Willebrand Factor

GPIb-IX

Glycoprotein Ib-IX Complex

P2X

Purinergic Channel X1

TRPC6

Transient Receptor Potential C6

ORAI1

Calcium Release-Activated Calcium Modulator 1

TxA2

Thromboxane A2

PAR1

Protease-Activated Receptor 1

P2Y12

Purinergic Receptor P2Y12

FDA

Food and Drug Administration

GPIIb/IIIa

Glycoprotein IIb/IIIa

Rac1

Ras-related C3 botulinum toxin substrate 1

PI3K

Phosphatidylinositol 3-Kinase

AKT

Protein Kinase B

XST

XueShuanTong

ADAMTS13

A Disintegrin and Metalloproteinase with Thrombospondin Motifs 13

GPIbα

Glycoprotein Ib Alpha

GPIb-IX-V

Glycoprotein Ib-IX-V Complex

GPIbβ

Glycoprotein Ib Beta

GPIX

Glycoprotein IX

GPV

Glycoprotein V

Col 1

Collagen 1

Ca²⁺

Calcium Ion

MKs

Megakarycoytes

FLNA

Filamin A

COTL1

Coactosin-like Protein 1

Src

Proto-oncogene Tyrosine-protein Kinase Src

Lyn

Tyrosine-protein Kinase Lyn

EC

Extended-Closed

EO

Extended-Open

GPVI

Glycoprotein VI

ITAM

Immunoreceptor Tyrosine-Based Activation Motif

PDI

Protein Disulfide Isomerase

ERp5

Endoplasmic Reticulum Protein 5

ERp57

Endoplasmic Reticulum Protein 57

α2β1

Integrin Alpha-2 Beta-1

CRP

Collagen-Related Peptide

GTPase

Guanosine Triphosphatase

LRRD

Leucine-rich repeat domain

MSD

Mechanosensitive domain