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. 2024 Nov 22;392(1):100012. doi: 10.1124/jpet.124.002149

Platelet spreading and clot retraction are regulated by 2 distinct αIIbβ3 outside-in signaling pathways

Arjit Nigam 1, Voddarahally N Manjuprasanna 2, Meghna U Naik 2, Ulhas P Naik 2,∗
PMCID: PMC13632521  PMID: 39893014

Abstract

Bidirectional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets, αIIbβ3 is in a low-affinity ligand binding state. However, on platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of phosphoinositide-3-kinase, phospholipase C, protein kinase C, and focal adhesion kinase strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading, whereas inhibition of Rac1, rho-associated, coiled-coil containing protein kinase, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases are required for platelet spreading and FAK activation but suppress clot retraction because their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment.

Significance Statement

Current antiplatelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel antiplatelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling are poorly understood. The work included in this article delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

Key words: Platelets, Clot retraction, Platelet spreading, Integrin αIIbβ3, Signaling

1. Introduction

It is well established that platelets play a crucial role in cardiovascular disease, which is the number one cause of death in humans. Current antiplatelet drugs have limited efficacy and inherent side-effects of bleeding. There is an unmet need for more effective and safer antiplatelet drugs. Recent efforts from several laboratories have indicated that modulating αIIbβ3 outside-in signaling might provide superior benefits in controlling thrombosis without compromising hemostasis (Adair et al, 2020; La et al, 2023; Fernández et al, 2024).

The primary function of platelets is to achieve hemostasis during blood vessel injury to prevent excessive blood loss. The circulating platelets are pushed toward the periphery of vessel by larger blood cells (primarily red blood cells) in flowing blood, ideally placing them close to the endothelium, thereby facilitating a fast response to vascular damage (Aarts et al, 1988). A vascular lesion results in denudation of the endothelium thereby exposing extracellular matrix (ECM) proteins such as collagen and von Willebrand factor to the flowing blood. The platelets sense this injury and get activated. Activated platelets spread on the exposed ECM and aggregate to form a clot containing trapped red blood cells and fibrin. The platelet rich clot thus formed retracts to allow remodeling of the endothelium.

Platelet integrin αIIbβ3 is a transmembrane heterodimer of an α- and a β-subunit. The αIIb subunit is megakaryocyte/platelet lineage specific. In platelets, the β3 subunit predominantly interacts with the αIIb subunit forming αIIbβ3 (or glycoprotein IIb/IIIa), which is the most abundant glycoprotein expressed on the platelet surface (∼80,000 copies/resting human platelet) (Wagner et al, 1996). The preferred ligand for αIIbβ3 is fibrinogen (Fg), although it also binds to fibronectin and von Willebrand factor (Naik and Parise, 1997). In resting platelets, αIIbβ3 exists in a low-affinity state incapable of binding to its soluble ligand. Engagement of platelet adhesion receptors (eg, GPIb-IX-V and GPVI-FcRγ) or G-protein coupled receptors (eg, P2Y1, P2Y12, PAR-1, and PAR-4) by their ligands initiates a signaling cascade inside the cell that brings about modulation in αIIbβ3 affinity (integrin activation) (Shattil, 1999). This signaling cascade responsible for affinity modulation of αIIbβ3 is referred to as inside-out signaling. Under physiologic settings, the signal originating from different agonist receptors act cooperatively leading to rise of intracellular Ca2+. This results in the activation of the downstream effector CalDAG-GEF1 that triggers the Rap1-RIAM axis leading to binding of talin and possibly kindlin to the cytoplasmic tail of the β3 subunit. This in turn results in unclasping of the membrane proximal complex of αIIb and β3 cytoplasmic tails triggering a conformational switch of αIIbβ3 molecule from low-affinity state to high-affinity ligand binding state. Fibrinogen binding to the activated αIIbβ3 molecule results in clustering of αIIbβ3 (Li et al, 2010) and triggers another signaling cascade across the activated αIIbβ3 (that goes into the platelet from outside) known as outside-in signaling (Shattil et al, 1998). It has been reported that the αIIbβ3 outside-in signaling regulates platelet spreading on Fg, clot retraction, granule secretion (Shattil and Newman, 2004) as well as thromboxane A2 (TxA2) generation (Jin et al, 2002). The 2 major outside-in signaling-dependent platelet responses—spreading on immobilized Fg and fibrin clot retraction—are thought to be regulated by a common signaling cascade and either of the 2 has been interchangeably used as an experimental readout for evaluating αIIbβ3 outside-in signaling. Following this paradigm, several molecules have been reported to regulate the αIIbβ3 outside-in signaling presumably by forming a complex at the αIIbβ3 cytoplasmic tails (Durrant et al, 2017).

Resting platelets can adhere to immobilized Fg without the need of αIIbβ3 affinity modulation. Adherent platelets spread by extending filopodia and lamellipodia similar to structures to form a monolayer (Brass, 2009) that covers a large area of exposed ECM at the site of vascular lesion. Fibrin clot formation and its subsequent retraction are important for thrombus stability and endothelial remodeling. The clot retraction process is regulated by αIIbβ3 outside-in signaling during which the αIIbβ3 anchored to the actin cytoskeleton transmit a contractile force to the extracellular fibrin polymers (Schoenwaelder et al, 2010). Although several signaling molecules have been identified to participate in outside-in signaling, the molecules required for platelet spreading (expansion) and clot retraction (contraction) has not been well understood. Here, we show that 2 distinct outside-in signaling pathways induced by αIIbβ3 independently regulate platelet spreading and clot retraction. We show that phosphoinositide-3-kinase (PI3K), phospholipase C (PLC), protein kinase C (PKC), and focal adhesion kinase (FAK) are common for both platelet spreading and clot retraction. Rho-associated, coiled-coil containing protein kinase (ROCK), Rac1, and mitogen-activated protein kinases (MAPKs) are required for clot retraction but dispensable for platelet spreading. Furthermore, we show that Src family kinases (SFKs) positively regulate platelet spreading, but negatively regulate clot retraction. Our results suggest that platelet spreading and clot retraction should not be used interchangeably as readout for outside-in signaling, particularly while evaluating transgenic mice or pharmacological agents.

2. Materials and methods

Human Fg and thrombin were obtained from Enzyme Research Laboratories. Pharmacological inhibitors such as U73221 (PLC inhibitor), Rö-31-8220 (PKC inhibitor), LY294002 (Wortmannin inhibitor), SB203580 (p38 inhibitor), Y-27632 (ROCK inhibitor), NSC23766 (Rac1-GEF), and PP2 (SFK inhibitor) used in this study were bought from CalBiochem (EMD Millipore). Bisindolymaleimide I was obtained from Sigma. FAK inhibitor TAE-226 was from Novartis Pharma. All the reagents used were purchased from Sigma and Thermo Fisher unless otherwise mentioned.

2.1. Preparation of human platelet rich plasma

Whole blood was drawn into 9:1 (v/v) 3.8% sodium citrate by venipuncture from healthy (of both gender and race), drug-free adults under informed consent. Approval was obtained from the institutional review boards at Thomas Jefferson University, Philadelphia, according to the Declaration of Helsinki. Briefly, a 25 mL aliquot of anticoagulated blood was transferred to a 50 mL centrifuge tube and was spun at 200g for 10 minutes. Subsequently, the platelet rich plasma (PRP) was isolated by carefully pipetting it out into a fresh tube without disturbing the packed white and red blood cells. Platelet poor plasma was prepared by centrifuging 1 mL of the residual blood at 800g for 10 minutes and isolating the plasma devoid of any cells.

2.2. Preparation of washed human platelets

Whole blood was drawn into 6:1 (v/v) acid/citrate/dextrose solution (pH 4.5) by venipuncture from healthy, drug-free adults. Prostaglandin E1 (100 nM) (Shock et al, 1999) that inhibits platelet activation was added to the PRP and centrifuged at 800g for 10 minutes. Resuspended platelets into Tyrode’s buffer were incubated at 37 °C for 10 minutes prior to spinning them down again at 800g for 10 minutes. The final platelet pellet was resuspended in Tyrode’s buffer and incubated at 37 °C for 30 minutes before using for the experiments.

3. TxA2 generation

Human PRP was pretreated with 200 μM aspirin for 30 minutes. Washed and resuspended in Tyrode’s buffer and stimulated with 0.1 U/mL of thrombin under stirring condition. Platelet aggregates were separated by centrifugation and the supernatant was analyzed for TxB2 (a stable analog of TxA2) using ELISA (Naik et al, 2014).

3.1. Platelet spreading on immobilized Fg and differential interference contrast microscopy

Pre-washed coverslips (22 × 40 mm) were coated with 250 μl of Fg (100 μg/mL) or bovine serum albumin (BSA; 3%) dissolved in 1× PBS and were incubated at 37 °C for 60 minutes in a humidify chamber. Following the incubation, the fluid was aspirated, and the coverslips were blocked by putting 250 μl of 0.5% heat inactivated BSA dissolved in 1× PBS and incubating them at 37 °C for another 60 minutes in similar humid conditions. Concomitantly, platelets were prepared for the experiment. Briefly, human PRP containing prostaglandin E1 (100 nM) and aspirin (1 mM) was incubated for 30 minutes at 37 °C prior to starting the washing procedure. Subsequently, the washed platelets were diluted to a final concentration of 1 × 107 platelets/mL using Tyrode’s buffer. Apyrase (1 U/mL) was then added to the platelet suspension and incubated for 10 minutes at room temperature (RT). Next, aspirin-/apyrase-treated platelets (0.6–0.8 × 106/mL) were aliquoted in prelabeled microcentrifuge tubes and a pharmacological inhibitor dissolved in DMSO was added to the corresponding tubes and incubated for an additional 20 minutes at RT. Thereafter, the platelets were added to the respective coverslips after removing the blocking buffer. Coverslips containing platelets were incubated for 45–60 minutes at 37 °C in the humid chamber. The process of spreading on the Fg-coated control coverslips was monitored every 15 minutes microscopically using Nikon inverted light microscope equipped with 40× objective lens. At the end of spreading, excess fluid and unbound platelets were gently washed with Tyrode’s buffer and then fixed with 250 μl of freshly prepared 4% paraformaldehyde for 20 minutes at RT. Last, after washing twice with 1× PBS the coverslips were mounted on a clean glass slide in PBS and the edges were sealed with nail polish to prevent from drying. Differential interference contrast (DIC) images showing morphology of the platelets on coverslips were captured using Zeiss Axioskop II light microscope using Zeiss Plan-Apochromat 100×/1.4NA oil immersion lens. The images were digitally recorded using Zeiss AxioVision software. For quantification, the surface area (in pixels) of all platelets in a particular field of view was calculated using NIH ImageJ software. Images were processed in Adobe Photoshop. Each spreading experiment was repeated at least 3 times.

3.2. Platelet-mediated fibrin clot retraction

Clot retraction assays were performed as described (Flevaris et al, 2009). Briefly, the platelet concentration in human PRP was adjusted to 3.5 × 108 platelets/mL using platelet poor plasma. It was further incubated in the presence or absence of aspirin (1 mM) at 37 °C for 30 minutes. Thereafter, the aspirin-treated platelet suspension was aliquoted in polypropylene tubes and a pharmacological inhibitor was added to corresponding tubes and incubated for an additional 20 minutes at RT. Treated or untreated platelets were transferred to borosilicate glass tube. Clot retraction was initiated by addition of thrombin (0.2 U/mL) to the platelet suspension and vortexing briefly at a very slow speed and left undisturbed at 37 °C. Images of these tubes were captured every 15 minutes using a point-and-shoot Nikon digital camera starting from time zero until 90–120 minutes. For quantification, the 2-dimensional surface area of retracting clots in each tube at the given time point was measured using ImageJ software and used to calculate percent clot retraction. Each experiment was repeated at least 3 times.

3.3. Statistical analysis

Statistical analysis of the data was performed using Student’s t test (mean ± SEM). One-way ANOVA was used for multiple comparisons. P ≤ .05 was regarded as statistically significant. Each experiment was repeated independently at least 3 times.

4. Results

4.1. Signaling molecules involved in platelet spreading on immobilized Fg

To evaluate the involvement of known signaling molecules in the process of outside-in signaling dependent platelet spreading, we allowed platelets to spread on immobilized Fg or BSA. It should be noted here that the “inside-out” signaling was blocked by the addition of aspirin (1 mM, inhibits TxA2 synthesis) and apyrase (1 U/mL, scavenges secreted ADP) to mimic true outside-in signaling scenario. Thrombin is a strong agonist that robustly activates TxA2 generation through inside-out signaling. To make sure aspirin treatment did inhibit TxA2 generation, we evaluated thrombin-induced TxA2 generation. As expected, pretreatment of platelets with aspirin (200 μM) completely abrogated TxA2 generation (Fig. 1). Resting human platelets, when exposed to immobilized BSA, they loosely attached and remained discoid. However, on immobilized Fg, they rapidly attached and become spiky by giving out numerous filopodia. The spiky platelets eventually spread by forming lamellipodia to attain a pancake-like morphology (Fig. 2, Ai and Aii). Pretreatment of platelets with 10 μM of each PI3K inhibitor, LY294002, and pan PLC inhibitor, U73221, which inhibits PLC-β and -γ isoforms, resulted in failure of filopodia and lamellipodia formation and maintained platelets in discoid morphology (Fig. 2, Aiii and Aiv). Both PLC and PI3K inhibition maintained the platelets in discoid morphology compared with fully spread pancake like morphology of the vehicle-treated control platelets. Interestingly, 10 μM pan PKC inhibitors bisindolymaleimide I or Rö-31-8220, as well as FAK inhibitor TAE-226, blocked lamellipodia formation without affecting filopodia formation, and as a result, platelets remained in spiky morphology (Fig. 2, Av–Avii). Quantification of the average surface area of platelets more than 3 independent experiments revealed that all inhibitors significantly blocked platelets spreading (Fig. 2B).

Fig. 1.

Fig. 1

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Aspirin treatment abrogates thrombin-induced TxA2 generation. Human platelets pretreated with or without 200 μM aspirin were stimulated with 0.1 U/mL of thrombin and supernatant were analyzed for TxB2 generation. Data for 4 independent experiments. Error bars represent SEM (∗∗∗∗P < .0001).

Fig. 2.

Fig. 2

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PI3K, PLC, PKC, and FAK are essential for spreading of platelet over immobilized Fg. DIC images of washed human platelets (Ai) exposed to BSA (control) or (Aii) 100 μg/mL of immobilized Fg in the presence or absence of indicated pharmacological inhibitor (Aiii–Avii). (B) Quantification of average surface area of platelet (pixels) from (A). Surface area of more than 300 platelets from more than 3 independent experiments was quantitated using ImageJ software and presented as the average surface area (in pixels). Error bars represents SEM (∗P < .05).

4.2. Signaling molecules involved in fibrin clot retraction

Because clot retraction also depends on outside-in signaling, we evaluated the effect of these inhibitors on fibrin clot retraction. Percent clot retraction at a given time point was compared with appropriate vehicle control. We found that effect of PLC inhibitor was more pronounced on clot retraction, compared with PI3K, PKC, and FAK inhibitors, because it completely abolished the retraction process, whereas others delayed clot retraction kinetics (Fig. 3, A and B). Thus, it can be concluded that PLC is essential for both platelet spreading and clot retraction whereas PI3K, PKC, and FAK, although not essential, positively influence these processes. Similar delay in clot retraction kinetics was also observed when p38, ROCK, and Rac1 were inhibited (Fig. 4). Interestingly, the effect of Rac1 inhibitor was significant only in the early stage of clot retraction (Fig. 4). These results suggest that p38, ROCK, and Rac1 positively regulate the process of clot retraction.

Fig. 3.

Fig. 3

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PI3K, PLC, PKC, and FAK positively regulate clot retraction. Human PRP was pretreated with or without indicated inhibitors and clotted using 0.2 U/mL of thrombin. Representative photographic images of retracted clots at the 45-minute time point are shown. (A) Representative clot retraction image of DMSO (control) and or in the presence of inhibitor as indicated. Quantification of percent clot retraction from A. Using PI3K inhibitor (Bi); Pan-PLC inhibitor (Bii); PKC inhibitor (Biii); FAK inhibitor (Biv) was performed using ImageJ as described in Materials and methods. Error bars represent SEM (∗P < .05 and ∗∗P < .01).

Fig. 4.

Fig. 4

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ROCK, p38-MAPK, and Rac1 regulate clot retraction. Human PRP was pretreated with or without indicated inhibitors and clotted using 0.2 U/mL of thrombin. Images of retracted clot shown above are representative images at 45 minutes for p38 and ROCK inhibitor treatment. For Rac1 inhibitor treatment, the image shown is at the 15-minute time point. Quantification of Aii and Aiii from Ai and Bii from Bi was performed using ImageJ as described in Materials and methods. Error bars represent SEM (∗P < .05 and ∗∗P < .01).

4.3. Rac1 and ROCK does not regulate platelet spreading on immobilized Fg

It is well known that Rac1 regulates lamellipodia formation whereas MAPKs and ROCK are known to regulate MLC, which is important for contractile function. We therefore evaluated the effect of Rac1, p38, and ROCK inhibitors on platelet spreading. To our surprise, none of the inhibitors had any effect on platelet spreading on immobilized Fg, suggesting that contractile function involving actomyosin is not required for platelets spreading (Fig. 5).

Fig. 5.

Fig. 5

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Rac1 and ROCK are not required for platelet spreading on immobilized Fg. (A) DIC images of washed human platelet spreading on immobilized Fg (100 μg/mL) in the presence or absence of indicated pharmacological inhibitor. (B) Quantification of average surface area of platelet (pixels) from (A). Surface area of more than 300 platelets from more than 3 independent experiments was quantitated using ImageJ software and presented as the average surface area (in pixels).

4.4. SFKs positively regulate platelet spreading but negatively regulate clot retraction

SFKs, particularly c-Src and Lyn, have been shown to play a significant role in outside-in signaling. Therefore, we evaluated the effect of inhibition of these kinases in platelet spreading and clot retraction. We used 10 μM PP2 to inhibit SFKs and assessed its effect on both platelets spreading on immobilized Fg and fibrin clot retraction. As expected, inhibition of SFKs abolished platelet spreading (Fig. 6A). Both filopodia formation and lamellipodia formation were blocked leaving platelet in discoid conformation suggesting that SFKs is essential for early steps of outside-in signaling. Interestingly, when evaluated the effect of PP2 on clot retraction, we found accelerated rate of clot retraction suggesting that SFKs are not required for clot retraction, and it negatively regulates this process (Fig. 6, B and C).

Fig. 6.

Fig. 6

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SFKs positively regulate platelet spreading but negatively regulate clot retraction. DIC images of human platelets spread on immobilized Fg in the presence of absence of PP2 (10 μM) (A). (B) Images of retracted clot of human PRP pretreated with or without PP2 (10 μM) at various time points as indicated.

Thus, we were able to categorize 4 classes of signaling molecules: (1) those that are essential for both platelet spreading and clot retraction such as PLCγ and PI3K; (2) those that are not essential but positively regulate both such as PKC and FAK; (3) those that only positively regulate clot retraction such as ROCK and Rac1; and (4) those that are indispensable for platelet spreading but negatively affect clot retraction such as SFKs.

5. Discussion

In the field of platelet biology, it is widely believed that the αIIbβ3 outside-in signaling regulates 2 process platelet spreading and clot retraction besides having some contribution in granule secretion and thromboxane A2 generation (Jin et al, 2002; Shattil and Newman, 2004; Liu et al, 2005). To avoid complications from inside-out signaling, we used apyrase (that scavenges secreted ADP) and aspirin (known to inhibit cyclooxygenases essential for TxA2 generation) to mimic conditions of true αIIbβ3 “outside-in” signaling without any positive feedback from the ADP or TxA2 signaling. The results presented here question the existing paradigm of outside-in signaling and provide evidence that the 2 processes (ie, platelet spreading on Fg and clot retraction) are regulated differently by the αIIbβ3 outside-in signaling (Fig. 7) (Flevaris et al, 2007).

Fig. 7.

Fig. 7

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Schematic representation of αIIbβ3 outside-in signaling pathways that regulate platelet spreading and clot retraction. Clot retraction pathway is shown on the right and the platelet spreading pathway is shown on the left. c-Src suppresses clot retraction that is relieved by calpain cleavage of β3 tail. SFK dependent activation of PI3K, PLCγ induce intracellular Ca2+ rise that support filopodia formation via Pyk2, lamellipodia formation via CIB1/FAK, and clot retraction via PKC and MAP kinase-dependent cytoskeletal rearrangement and actomyosin activation.

Inhibition of SFKs abolishes Syk phosphorylation and its subsequent activation (Obergfell et al, 2002). During αIIbβ3 outside-in signaling, Src and Syk kinases are responsible for phosphorylation of tyrosine residues (Y700, Y731, and Y774) on adaptor protein c-Cbl (Buitrago et al, 2011). Y731 phosphorylated c-Cbl provides a docking site for SH2-domain containing p85 subunit of PI3K (Hunter et al, 1999). It is reported that PI3K functions upstream of PLCγ2 (Dai et al, 2006), which hydrolyzes phosphatidylinositol 4,5-bisphosphate generating inositol 1,4,5-triphosphate and diacylglycerol. Release of calcium ions from internal stores on binding of inositol 1,4,5-triphosphate to its receptors together with diacylglycerol aids in activation of PKCs. Active PKCs have been implicated in phosphorylation of various cytoskeletal proteins that regulate cytoskeletal rearrangement. Recently, we have shown that dynamic cytoskeletal rearrangement is important for FAK phosphorylation at Y397 and its activation (Fig. 7) (Naik et al, 2017).

Our finding that pretreatment with pan-PLC inhibitor (10 μM U73122) resulted in the inability of platelets to form filopodia or lamellipodia on immobilized Fg is consistent with the findings of Wonerow et al (2003) who also saw similar inhibition in platelet spreading in the presence of PLC inhibitor. Furthermore, pharmacological inhibition of PI3K, also showed failure to form filopodia and lamellipodia compared with the vehicle-treated control platelets is consistent with a previously published report where the authors also found inhibition of platelet spreading on inhibition of PI3K (Heraud et al, 1998). Interestingly, treatment of platelets with inhibitors of PKC and FAK resulted in spiky morphology of platelets with limited to no lamellipodia formation, which is consistent with results observed by Haimovich et al (1996) and Wonerow et al (2003) as well as by Jones et al (2009) who used structurally distinct FAK inhibitor PF-573,228. Besides such pharmacological approaches, reports from Buensuceso et al (2005), Soriani et al (2006), and Chaudhary et al (2020) using genetic approaches demonstrated that the β, θ, and the δ isoforms of PKC are of particular importance for murine platelet spreading on immobilized Fg. Hitchcock et al (2008) found significantly less surface area in FAK−/− platelets as compared with wild-type platelets on spreading on Fg. Similarly, genetic ablation of Cib1, which is known to activate FAK, also results in defective spreading without affecting filopodia formation (Naik et al, 2009, 2017). The ability of platelets to form filopodia under such conditions could be due to the ability of Ca2+ to activate Pyk2, a Ca2+-sensitive family member of FAK (Ren et al, 2001).

Our finding that PI3K inhibition resulted in delayed clot retraction is in agreement with Schoenwaelder et al (2010) who reported similar initial lag in the initiation of clot retraction and demonstrated that it is the type 1a p110-β isoform of PI3K that plays the major role in regulating fibrin clot retraction in ex vivo conditions. Akt, a downstream effector of PI3K pathway, does not seem to be important for platelet spreading because its activation is dependent on secreted ADP (O’Brien et al, 2012). Suzuki-Inoue et al (2007), using PLCγ2−/− platelets, demonstrated that deletion of this isoform of PLC, which is reported to play a role downstream of the integrin αIIbβ3 outside-in signaling, resulted in delayed but, did not prevented clot retraction from reaching completion probably due to intact PLC-β isoform. The complete blockade of clot retraction by pan PLC inhibitor in our study can be explained by inhibition of both PLCγ and PLC-β which functions downstream of thrombin receptors.

It was observed that both p38-MAPK, Rac1, and ROCK inhibitors resulted in delayed clot retraction without affecting platelet spreading suggesting that they are only involved in clot retraction. These results are consistent with those published by Flevaris et al (2009) and Gong et al (2010). Contrary to these results, Aslan et al (2011) saw a marked reduction in platelet spreading upon inhibition of Rac1 with 2 structurally distinct Rac1 inhibitors (300 μM NSC23766 and 50 μM EHT1864), probably due to high concentrations of inhibitors used.

Platelets treated with SFK inhibitor failed completely to spread on immobilized Fg that is consistent with results reported by Wonerow et al (2003) and Aslan et al (2011). However, the same concentration of SFK inhibitor resulted in enhanced clot retraction that is also observed by Flevaris et al (2009). The enhanced clot retraction can be explained by the fact that SFKs activate RhoGAP that is known to inhibit RhoA, an activator of ROCK that positively regulates MLC phosphorylation via inhibition of MLC phosphatase (Gong et al, 2010; Pleines et al, 2012). Furthermore, increased Ca2+ in addition to inducing CIB1, a Ca2+-binding protein, dependent FAK activation (Naik and Naik, 2003; Naik et al, 2017), also causes a late event of β3 tail cleavage by calpain, resulting in a loss of Src from β3, thus promoting clot retraction process (Flevaris et al, 2007; Gong et al, 2010).

Mechanistically, when platelets are exposed to immobilized Fg, multiple αIIbβ3 molecules bind to Fg underside of the spreading platelets (2-dimensional) causing a lateral expansion force to achieve spreading. On the other hand, during clot retraction process αIIbβ3 binds to fibrin in a 3-dimensional space and causes a contractile force through its interaction with cytoskeleton. Thus, it is conservable that the 2 events could be regulated by 2 distinct signaling pathways. Most of the studies aimed at identifying signaling molecules involved in αIIbβ3 outside-in signaling rely on platelets exposed to immobilized Fg, a condition conducive to platelet spreading. A clot retracting event may involve distinct signaling molecules in addition to those involved in platelet spreading. Furthermore, in addition to integrin outside-in signaling, signaling through PARs, thrombin receptors, and GPVI, which is recently has been shown to bind fibrin, may participate in clot retraction but not in platelet spreading. Therefore, caution must be used in interpreting the involvement of signaling molecules in outside-in signaling.

Conflict of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors thank Kushal U. Naik for editing the manuscript. This work was submitted as part of an MS thesis by Arjit Nigam (UDspace.udel.edu).

Financial support

This work was funded by National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01HL113188, R01HL119374, and R01HL143959] (to U.P.N.).

Data availability

This article contains no datasets generated or analyzed during the current study.

Authorship contributions

Participated in research design: Nigam, M.U. Naik, U.P. Naik.

Conducted experiments: Nigam, Manjuprasanna, M.U. Naik.

Performed data analysis: Nigam, M.U. Naik, U.P. Naik.

Wrote or contributed to the writing of the manuscript: M.U. Naik, U.P. Naik.

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Associated Data

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Data Availability Statement

This article contains no datasets generated or analyzed during the current study.

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