The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation

Abstract LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.

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PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets

Thrombosis and Haemostasis ◽

10.1160/th07-03-0207 ◽

2007 ◽

Vol 98 (10) ◽

pp. 806-812 ◽

Cited By ~ 46

Author(s):

Vandana Dole ◽

Wolfgang Bergmeier ◽

Ian Patten ◽

Junichi Hirahashi ◽

Tanya Mayadas ◽

...

Keyword(s):

Endothelial Activation ◽

Leukocyte Rolling ◽

Wild Type ◽

Platelet Surface ◽

Activated Platelets ◽

And Function ◽

Body Secretion

SummaryWe have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1-/- compared to wild-type mice. The leukocyte integrin αMβ2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte- neutrophil elastase,known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.

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Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation

Thrombosis and Haemostasis ◽

10.1160/th03-03-0145 ◽

2003 ◽

Vol 90 (10) ◽

pp. 672-678 ◽

Cited By ~ 17

Author(s):

Zhang Jian-ning ◽

Angela Bergeron ◽

Qinghua Yu ◽

Carol Sun ◽

Latresha McBride ◽

...

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Platelet Activation ◽

Whole Blood ◽

Fluid Shear Stress ◽

High Shear ◽

Platelet Response ◽

Short Pulses ◽

Hydrodynamic Effects

SummaryPlatelet functions are increasingly measured under flow conditions to account for blood hydrodynamic effects. Typically, these studies involve exposing platelets to high shear stress for periods significantly longer than would occur in vivo. In the current study, we demonstrate that the platelet response to high shear depends on the duration of shear exposure. In response to a 100 dyn/cm2 shear stress for periods less than 10-20 sec, platelets in PRP or washed platelets were aggregated, but minimally activated as demonstrated by P-selectin expression and binding of the activation-dependent αIIbβ3 antibody PAC-1 to sheared platelets. Furthermore, platelet aggregation under such short pulses of high shear was subjected to rapid disaggregation. The disaggregated platelets could be re-aggregated by ADP in a pattern similar to unsheared platelets. In comparison, platelets that are exposed to high shear for longer than 20 sec are activated and aggregated irreversibly. In contrast, platelet activation and aggregation were significantly greater in whole blood with significantly less disaggregation. The enhancement is likely via increased collision frequency of platelet-platelet interaction and duration of platelet-platelet association due to high cell density. It may also be attributed to the ADP release from other cells such as red blood cells because increased platelet aggregation in whole blood was partially inhibited by ADP blockage. These studies demonstrate that platelets have a higher threshold for shear stress than previously believed. In a pathologically relevant timeframe, high shear alone is likely to be insufficient in inducing platelet activation and aggregation, but acts synergistically with other stimuli.

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Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

Journal of Experimental Medicine ◽

10.1084/jem.20071800 ◽

2007 ◽

Vol 204 (13) ◽

pp. 3103-3111 ◽

Cited By ~ 188

Author(s):

Brian G. Petrich ◽

Patrizia Marchese ◽

Zaverio M. Ruggeri ◽

Saskia Spiess ◽

Rachel A.M. Weichert ◽

...

Keyword(s):

Platelet Adhesion ◽

Ex Vivo ◽

Focal Adhesions ◽

Β1 Integrin ◽

Normal Morphology ◽

And Function ◽

Specific Deletion

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

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The N-Terminus ofDictyosteliumScar Interacts with Abi and HSPC300 and Is Essential for Proper Regulation and Function

Molecular Biology of the Cell ◽

10.1091/mbc.e06-06-0518 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1609-1620 ◽

Cited By ~ 12

Author(s):

Diana Caracino ◽

Cheryl Jones ◽

Mark Compton ◽

Charles L. Saxe

Keyword(s):

Actin Polymerization ◽

Terminal Region ◽

Wild Type ◽

Large Molecular Weight ◽

Weight Protein ◽

And Function ◽

Necessary And Sufficient

Scar/WAVE proteins, members of the conserved Wiskott-Aldrich syndrome (WAS) family, promote actin polymerization by activating the Arp2/3 complex. A number of proteins, including a complex containing Nap1, PIR121, Abi1/2, and HSPC300, interact with Scar/WAVE, though the role of this complex in regulating Scar function remains unclear. Here we identify a short N-terminal region of Dictyostelium Scar that is necessary and sufficient for interaction with HSPC300 and Abi in vitro. Cells expressing Scar lacking this N-terminal region show abnormalities in F-actin distribution, cell morphology, movement, and cytokinesis. This is true even in the presence of wild-type Scar. The data suggest that the first 96 amino acids of Scar are necessary for participation in a large-molecular-weight protein complex, and that this Scar-containing complex is responsible for the proper localization and regulation of Scar. The presence of mis-regulated or unregulated Scar has significant deleterious effects on cells and may explain the need to keep Scar activity tightly controlled in vivo either by assembly in a complex or by rapid degradation.

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P-Selectin and Platelet Clearance

Blood ◽

10.1182/blood.v92.11.4446.423k19_4446_4452 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4446-4452 ◽

Cited By ~ 3

Author(s):

Gaëtan Berger ◽

Daqing W. Hartwell ◽

Denisa D. Wagner

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Platelet Activation ◽

Soluble Form ◽

Wild Type ◽

Adhesion Receptor ◽

Activated Platelets ◽

Deficient Mice

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

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Platelet JNK1 is involved in secretion and thrombus formation

Blood ◽

10.1182/blood-2009-07-233932 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4083-4092 ◽

Cited By ~ 72

Author(s):

Frédéric Adam ◽

Alexandre Kauskot ◽

Paquita Nurden ◽

Eric Sulpice ◽

Marc F. Hoylaerts ◽

...

Keyword(s):

Platelet Aggregation ◽

Thrombus Formation ◽

Blood Perfusion ◽

Collagen Matrix ◽

In Vivo Model ◽

Wild Type ◽

Platelet Secretion

Abstract The role of c-Jun NH2-terminal kinase 1 (JNK1) in hemostasis and thrombosis remains unclear. We show here, with JNK1-deficient (JNK1−/−) mice, that JNK1 plays an important role in platelet biology and thrombus formation. In tail-bleeding assays, JNK1−/− mice exhibited longer bleeding times than wild-type mice (396 ± 39 seconds vs 245 ± 32 seconds). We also carried out in vitro whole-blood perfusion assays on a collagen matrix under arterial shear conditions. Thrombus formation was significantly reduced for JNK1−/− platelets (51%). In an in vivo model of thrombosis induced by photochemical injury to cecum vessels, occlusion times were 4.3 times longer in JNK1−/− arterioles than in wild-type arterioles. Moreover, in vitro studies carried out in platelet aggregation conditions demonstrated that, at low doses of agonists, platelet secretion was impaired in JNK1−/− platelets, leading to altered integrin αIIbβ3 activation and reduced platelet aggregation, via a mechanism involving protein kinase C. JNK1 thus appears to be essential for platelet secretion in vitro, consistent with its role in thrombus growth in vivo. Finally, we showed that ERK2 and another isoform of JNK affect platelet aggregation through 2 pathways, one dependent and another independent of JNK1.

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MiR-223 is dispensable for platelet production and function in mice

Thrombosis and Haemostasis ◽

10.1160/th13-07-0623 ◽

2013 ◽

Vol 110 (12) ◽

pp. 1207-1214 ◽

Cited By ~ 14

Author(s):

Xavier Loyer ◽

Simon Leierseder ◽

Tobias Petzold ◽

Lin Zhang ◽

Steffen Massberg ◽

...

Keyword(s):

Platelet Adhesion ◽

Cell Types ◽

Surface Expression ◽

Wild Type ◽

Platelet Production ◽

Multiple Cell ◽

And Function ◽

Deficient Mice ◽

Platelet Depletion

SummaryMicroRNAs (miRNAs) are key physiological regulators in multiple cell types. Here, we assessed platelet production and function in mice deficient in miR-223, one of the most abundantly expressed miRNAs in platelets and megakaryocytes. We found platelet number, size, lifespan as well as surface expression of platelet adhesion receptors to be unchanged in miR-223-deficient mice. Likewise, loss of miR-223 did not affect platelet activation, adhesion and aggregation and also had no effect on bleeding times. Moreover, miR-223 null megakaryocytes developed normally and were capable to form pro-platelets. However, we detected a transient delay in the recovery of platelet numbers following antibody-induced platelet depletion in miR-223-deficient animals. This delay was not observed after transplantation of bone marrow from miR-223-deficient animals into wild-type recipients, indicating a non-cell-autonomous role of miR-223 for thrombopoiesis. Overall, our data indicate a surprisingly modest role of miR-223 in platelet production, while the function of platelets does not seem to depend on miR-223.

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The Role of the cGMP-Dependent Protein Kinase II in Platelet Activation and In Vivo Thrombosis.

Blood ◽

10.1182/blood.v106.11.653.653 ◽

2005 ◽

Vol 106 (11) ◽

pp. 653-653

Author(s):

Zhenyu Li ◽

Guoying Zhang ◽

Hong Yin ◽

Robert Feil ◽

Franz Hofmann ◽

...

Keyword(s):

Platelet Activation ◽

Low Dose ◽

Bleeding Time ◽

Wild Type ◽

Rt Pcr ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

Abstract Although it was previously believed that the intracellular secondary messenger cGMP inhibits platelets, we have recently shown that cGMP-dependent protein kinase I (PKG I) in fact plays a stimulatory role in platelet activation. However, there are apparent differences between the PKG inhibitors and PKG I knockout in their effects on platelet activation. PKG inhibitors are more potent in inhibiting platelet activation than PKG I knockout. More importantly, although platelet secretion and aggregation induced by collagen were inhibited by PKG inhibitors, they are not significantly affected in PKG I knockout platelets. There are two types of PKG, PKG I and PKG II. PKG II has not been previously described in platelets. Here we show that PKG II mRNA is expressed in platelets using RT-PCR with primers specific for a C-terminal fragment of human PKG II cDNA. We further cloned the complete cDNA of human PKG II by RT-PCR using the purified human platelet mRNA as a template. Furthermore, PKG II from platelet lysates was pulled down by cGMP conjugated agarose beads and detected by western blot using a polyclonal antibody against PKG II. These data indicate that PKG II is expressed in platelets. To investigate the role of PKG II in platelet activation, washed wild type or PKG II knockout (PKG II−/−) mouse platelets in tyrode’s solution were exposed to platelet agonists. Platelet aggregation and ATP secretion induced by low concentrations of collagen were significantly reduced in PKG II deficient mice, indicating that PKG II plays important roles in collagen-induced platelet activation. PKG II−/− platelets also showed reduced aggregation and secretion to low dose of a thromboxane A2 (TXA2) analog, U46619. However, low dose thrombin-induced platelet activation was not negatively affected in PKG II−/− platelets, but was inhibited in PKG I−/− platelets. To evaluate the in vivo role of PKG II, we compared in vivo thrombus formation of wild type and PKG II knockout mice using the FeCl3-injured carotid artery thrombosis model. The time to the formation of stable thrombus in PKG II−/− mice (median, 420.0 seconds, n=15) is significantly prolonged compared to wild type mice (median, 321.0 seconds, n=15) (p=0.031). Tail-bleed time analysis also indicated a remarkably prolonged bleeding time in PKG II−/− mice (the median bleeding time was 73.50 seconds (n=18) in wild type mice, 454.50 seconds (n=20) in PKG II knockout mice) (p=0.0008). Thus, PKG II plays an important role in promoting platelet activation, thrombosis and hemostasis. PKG I and PKG II have differential roles in platelet activation induced by different platelet agonists.

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Knockout of the PKC Substrate Pleckstrin Causes Pleomorphic Defects in Platelets, Lymphocytes and Granulocytes.

Blood ◽

10.1182/blood.v108.11.394.394 ◽

2006 ◽

Vol 108 (11) ◽

pp. 394-394

Author(s):

Lurong Lian ◽

Yanfeng Wang ◽

Xinsheng Chen ◽

Tami Bach ◽

Laurie Lenox ◽

...

Keyword(s):

T Cells ◽

Platelet Aggregation ◽

Alternative Pathway ◽

Second Messengers ◽

Pi3k Inhibitor ◽

Loss Of Function ◽

Wild Type

Abstract Pleckstrin is a 40 kDa phosphoprotein containing amino- and carboxyl-terminal Pleckstrin Homology (PH) domains separated by a DEP domain. Pleckstrin’s expression is restricted to platelets and leukocytes, and represents approximately 1% of total cellular protein within these cells. Following platelet and leukocyte activation, PKC rapidly phosphorylates pleckstrin inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Heterologously expressed phosphorylated pleckstrin colocalized with integrins and induces cytoskeletal reorganization. To better define the role of pleckstrin in vivo, we introduced a loss-of-function mutation into the murine pleckstrin gene. Pleckstrin-null mice were present in offspring at a frequency consistent with a Mendelian inheritance pattern. Adult pleckstrin −/− mice had 32% lower platelet counts than their littermates, but exhibited no spontaneous hemorrhage. Given the role of PKC and phospholipid second messengers on cytoskeletal dynamics, and our observations of pleckstrin overexpression in cell lines, we analyzed whether loss of pleckstrin affected cell spreading. Pleckstrin −/− platelets spread extremely poorly upon immobilized fibrinogen, and rarely exhibited broad membrane extensions. Granulocytes from pleckstrin −/− mice also have a spreading defect, as well as impaired ability to generate reactive oxygen species in the response to TNFα. Knockout B-cells, CD4-T-cells, and CD8-T-cells all migrated approximately 30% as efficiently as wild type cells in response to a gradient of SDF-1α in a transwell assay. These data suggest that loss of pleckstrin causes cytoskeletal defects in cells of multiple hematopoietic lineages. Analyzing whether this caused a functional defect, we found that pleckstrin −/− platelets exhibited a 22% dense- and 24% alpha-granule exocytosis defect, and a 35% defect in thrombin-induced calcium entry. In spite of these abnormalities, platelets changed shape and aggregated normally after stimulation with thrombin, ADP, or collagen in vitro. Pleckstrin knockout platelets did have a markedly impaired aggregation response following exposure to the PKC stimulant, PMA. This suggested that pleckstrin is a critical effector for PKC-mediated aggregation, but another pathway is able to compensate for this loss of pleckstrin following agonist stimulation. We reasoned that the alternative pathway might also utilize PIP2-dependent second messengers. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet aggregation, we tested whether PI3K compensated for the loss of pleckstrin. We found that the PI3K inhibitor, LY294002 profoundly impaired the aggregation of pleckstrin knockout platelets in response to stimulation of the thrombin receptor. In contrast, the PI3K inhibitor minimally affected wild type platelets. This demonstrates that second messengers generated by PI3K are able to compensate for loss of pleckstrin. This also demonstrates that thrombin-induced platelet aggregation can be mediated by one of two parallel pathways, one involving PKC and pleckstrin, and the other involving PI3K. Together, our results show that pleckstrin is an essential component of PKC-mediated platelet activation and signals directed to the cytoskeleton.

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Biphasic Roles for the Soluble Guanylyl Cyclase (sGC) In Platelet Activation In Mice

Blood ◽

10.1182/blood.v116.21.486.486 ◽

2010 ◽

Vol 116 (21) ◽

pp. 486-486

Author(s):

Guoying Zhang ◽

Binggang Xiang ◽

Radek C. Skoda ◽

Susan S. Smyth ◽

Xiaoping Du ◽

...

Keyword(s):

Platelet Activation ◽

Guanylyl Cyclase ◽

Conflicts Of Interest ◽

Soluble Guanylyl Cyclase ◽

Wild Type ◽

No Donor ◽

Cgmp Production ◽

Deficient Mice

Abstract Abstract 486 The role of intracellular secondary messenger cGMP in platelet activation has been controversial, with both stimulatory and inhibitory roles reported. The platelet cGMP is believed to be predominantly synthesized by soluble guanylyl cyclase (sGC), which is activated by nitric oxide (NO). To specifically determine the role of sGC-dependent cGMP synthesis in platelet function and in vivo thrombosis and hemostasis, we produced mice harboring a “floxed” sGC beta1 allele. In the “floxed” sGC beta1 mice (sGC beta1fl/fl), the exons 7 and 8 of sGC beta1 gene and an inserted Neo cassette were flanked with three LoxP sites. Platelet-specific deletion of sGC beta1fl/fl allele was accomplished through breeding of the sGC beta1fl/fl mice with pf4-Cre recombinase transgenic mice. Immunoblotting showed the complete absence of this protein in sGC beta1fl/fl/Cre platelets. Mice lacking sGC beta1 in platelets appeared to develop normally and had normal blood counts, including platelets. Blood pressure of platelet-specific sGC deficient mice was comparable to that of wild-type littermates. Inactivating the sGC beta1 gene in platelets abolished cGMP production induced by either NO donors or platelet agonists that are known to activate endogenous NO synthesis, confirming that both the platelet agonist-induced and NO donor-induced platelet cGMP production are predominantly mediated by sGC. Platelets lacking sGC exhibit a marked defect in aggregation and secretion in response to low doses of platelet agonists, collagen and thrombin. Importantly, tail-bleeding times were significantly prolonged in the platelet-specific sGC deficient mice compared with the wild-type littermates. In a FeCl3-induced carotid artery thrombosis model, time to occlusive thrombosis was prolonged in the platelet-specific sGC deficient mice, compared to wild type littermates. Thus, the agonist-stimulated sGC activation is important in promoting platelet granule secretion and aggregation. On the other hand, NO donor SNP-induced inhibition of platelet activation was abolished in sGC-deficient platelets. However, at high concentrations (>100μM), SNP inhibited platelet activation in both wild type and sGC deficient mice, indicating that both cGMP-dependent and -independent mechanisms are involved in NO donor-induced inhibition of platelet activation. Together, our data demonstrate that sGC contributes to both agonist-induced platelet activation and NO donor-induced platelet inhibition. Disclosures: No relevant conflicts of interest to declare.

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