On account of the ability to form intravascular thrombus following the erosion or rupture of atherosclerotic plaques, platelets are known to play an essential role in the pathophysiology of IS [14]. Platelet parameters like MPV and PLT count are considered as significant determinants of platelet function [29]. There is evidence that increased platelet size and count reflects increased platelet activity and are useful predictive and prognostic biomarkers for cerebrovascular events. In a recent study published from our lab an elevated MPV was found to be significantly associated with increased risk of IS and also higher clot rate and higher degree of disability based on mRS. [15]. These results are in accordance with previous studies where an increase in MPV has been associated significantly with increased risk of IS [12, 29–31]. In the current study we screened the genetic variants involved in PLT count, MPV and platelet reactivity in IS patients. Based on the previous reports a total of 106 variants in 96 genes involved in PLT count, MPV and Platelet reactivity were initially screened using GSA in 17% patients. Out of these, 62 variants have been reported to affect PLT count; 33 were found to affect MPV and 11 variants reported to affect platelet reactivity (Tables 10, 11 and 12). Most of these genes were found to be either normal homozygous or showed very minor frequency of heterozygosity except for variants of two genes ARHGEF3 (rs1354034, T > C), and THPO (rs6141, C > T). Therefore, these were evaluated further by Sanger Sequencing after amplifying the specific regions of these genes bearing the variation in all the subjects.
Table 11
SNPs associated with PLT Count
SNP | Gene | SNP | Gene |
rs2336384 | MFN2 | rs373121156 | CDKN2A |
rs10914144 | DNM3 | rs117899880 | BRD3 |
rs1668871 | TMCC2 | rs505404 | PSMD13 |
rs7550918 | LOC148824 | rs4246215 | FEN1 |
rs3811444 | TRIM58 | rs4938642 | CBL |
rs12603268 | GCKR | rs7342306 | CD9-VWF |
rs625132 | EHD3 | rs941207 | BAZ2A |
rs17030845 | THADA | rs3184504 | SH2B3 |
rs76160061 | SYN2 | rs17824620 | RPH3A-PTPN11 |
rs7641175 | SATB1 | rs7961894 | WDR66 |
rs1354034 | ARHGEF3 | rs4148441 | ABCC4 |
rs3792366 | PDIA5 | rs8022206 | RAD51L1 |
rs7694379 | HSD17B13 | rs8006385 | ITPK1 |
rs17568628 | F2R | rs7149242 | C14orf70-DLK1 |
rs700585 | MEF2C | rs11628318 | RCOR1 |
rs2070729 | IRF1 | rs2297067 | C14orf73 |
rs441460 | LRRC16 | rs3809566 | TPM1 |
rs3819299 | HLA-B | rs1719271 | ANKDD1A |
rs399604 | HLA-DOA | rs6065 | GP1BA |
rs210134 | BAK1 | rs397969 | AKAP10 |
rs9399137 | HBS1L-MYB | rs55997232 | TAOK1 |
rs342275 | PIK3CG | rs10512472 | SNORD7-AP2B1 |
rs4731120 | WASL | rs708382 | FAM171A2-ITGA2B |
rs6995402 | PLEC1 | rs11082304 | CABLES1 |
rs409801 | AK3 | rs8109288 | TMP4 |
rs13300663 | RCL1 | rs17356664 | EXOC3L2 |
rs1034566 | ARVCF | rs12526480 | LRRC16A |
rs6141 | THPOII | rs6490294 | ACAD10 |
rs9494145 | HBS1L-MYB | rs477895 | BAD |
rs7896518 | JMD1C | rs13236689 | CD36 |
rs151361 | LRRC16A | rs342293 | PIK3CG |
Table 12
SNP | Gene | SNP | Gene |
rs7961894 | WDR66 | rs10512627 | KALRN |
rs8109288 | TPM4 | rs117341321 | KIAA0232 |
rs1354034 | ARHGEF3 | rs2227831 | F2R |
rs342293 | PIK3CG | rs4521516 | MEF2C |
rs7075195 | JMD1C | rs10076782 | RNF145 |
rs8076739 | TAOK1 | rs10813766 | DOCK8 |
rs117213068 | TMCC2 | rs7075195 | JMJD1C |
rs17655730 | PSMD13 | rs17655730 | NLRP6 |
rs4812048 | CTSZ-TUBB1 | rs1558324 | CD9-VWF |
rs342296 | PIK3CG | rs2015599 | MTSTD1 |
rs11653144 | TAOK1 | rs10876550 | COPZ1-NFE2-CBX5 |
rs17396340 | KIF1B | rs2950390 | PTGES3 |
rs10914144 | DNM3 | rs7317038 | GRTP1 |
rs649729 | EHD3 | rs944002 | C14orf73 |
rs4305276 | ANKMY1 | rs3000073 | BRF1 |
rs1354034 | ARHGEF3 | rs16971217 | AP2B1 |
rs12969657 | CD226 | | |
Table 13
SNPs associated with PLT Reactivity
SNP | Gene |
rs1613662 | GP6 |
rs3557 | FCER1G |
rs3737224 | PEAR1 |
rs11264579 | PEAR1 |
rs3729931 | RAF1 |
rs147212241 | P2RY12 |
rs3788337 | GNAZ |
rs10496541 | CD36 |
rs35091628 | MAP2K2 |
rs12566888 | PEAR1 |
rs7940646 | MRVI1 |
Thrombopoietin (also known as THPO, TPO) is a major cytokine that plays a crucial role in platelet production. This humoral substance controls MK proliferation and differentiation to maintain normal thrombopoiesis [32]. The SNP rs6141 (C > T) situated at 3’-UTR region of the THPO gene is reported to be involved in the post transcriptional control of the gene expression mainly affecting mRNA splicing [33]. Studies have also shown that microdeletions involving this SNP in THPO gene cause mild congenital thrombocytopenia [34, 35]. Further two gain-of-function mutations in THPO gene G > C transversion and a G > T transversion have been reported to produce mRNAs with shortened 5′–untranslated regions (UTR) that are more efficiently translated in comparison with transcripts produced by wild type THPO. These transcripts with gain of function mutation result in elevated PLT count which might lead to thrombosis and bleeding [36, 37]. THPO variants with bi-allelic loss-of-function cause multilineage bone marrow failure and severely reduced platelet counts [38–40]. Another study identified a one-base deletion in the 5′-untranslated region of the THPO gene. In vitro experiments showed that this mutation increased TPO production and suggested that this region of the THPO gene may play a crucial role in regulating THPO expression [41]. Based on the results of different GWAS studies it has been established that rs6141 of THPO gene is a significant determinant of MPV and PLT count [33, 42, 43]. As far as the role of THPO in IS is concerned, it has been reported that elevated levels of TPO are associated with increased MPV and PLT counts in these patients [44–46].
In the current study evaluating the association of rs6141 (THPO) with IS we found a significant association of TT genotype with the disease which was confirmed by MLR analysis showing an independent association of TT genotype with the disease (p < 0.05). However, we did not find a significant difference in the distribution of T and C alleles between patients and controls. As far as the association of this variant with IS subtypes is concerned, the T allele showed a significant association with LAA, Small artery occlusion, and cardioembolism. We also evaluated the association of variant genotype with MPV, Clot rate, and PLT count. The TT and CT genotypes showed a significant association with elevated MPV, higher clot rate and reduced PLT count in comparison with the CC genotype bearing patients. This association was also confirmed by MLR controlling all other confounding factors.
Since the focus of the current study was on platelet parameters, therefore, the expression analysis of THPO (rs6141) was carried out using mRNA isolated from platelets of IS patients. Patients bearing TT genotype showed highest expression followed by CT and CC genotypes. Aged platelets induce the production of TPO in the hepatocytes. TPO increases the number of circulating platelets once released into the bloodstream. Most of the studies have linked higher TPO levels with increased platelet activity [46]. A study carried out by Balcik et al (2013) reported that patients with IS have higher TPO and MPV levels and concluded that increased TPO levels elevate both PLT count and MPV resulting in higher thrombotic capacity of platelets [44]. Another study carried out in acute myocardial infarction (AMI) patients and unstable angina pectoris also reported that increased TPO and MPV levels are positively associated with each other in AMI patients [47]. Yang et al (2008) evaluated the role of Severe Acute Respiratory Syndrome (SARS) in affecting normal functions of hematopoietic stem cells and megakaryocytic cells. They found that increased TPO levels in the plasma of these patients lead to thrombocytosis and hyperactive platelets [48]. Recently a study demonstrated that platelets in COVID-19 patients aggregate faster and showed increased spreading on both fibrinogen and collagen during clot retraction. It was also found that TPO levels were elevated in the serum of SARS-CoV-2 patients [49].
Previous studies have mostly explored the association of THPO with PLT count. However, its role in MPV has not been studied much. Since MPV and PLT count are inversely correlated therefore it is obvious that studies showing its association with increased PLT count might not have observed its impact on MPV [50].
The proposed mechanism by which TT genotype (rs6141) might lead to the higher expression of THPO gene in bone marrow producing hyperactive platelets has been depicted in Fig. 14. TPO binds to the megakaryocytes or platelets and controls their production through a feedback mechanism. During normal hemostasis TPO concentrations remain normal. Platelets experience mechanical stress that shortens their life span under the conditions like atherosclerosis which indirectly activates platelet biogenesis [51] [44]. In vivo injection of recombinant adenoviral vectors, or transgenes resulted in variable thrombocytosis [52–55]. In addition, studies have also demonstrated that patients bearing gain of function mutations in THPO gene, enhance TPO mRNA translation which elevates its expression inducing lineage-selective effects in patients affected with thrombocytosis and polyclonal hematopoiesis. TPO levels were also observed to be higher in the serum [37, 41, 56]; [57]. It has been reported that around 1000 to 3000 platelets are produced from a single MK [58]. The mechanism leading to the production of platelets through MKs involves lot of reorganization of cytoskeletal components like actin and tubulin. THPO is the major watch dog of this process [59, 60]. Although the process of thrombopoiesis is understood well there are still many unanswered questions to how some transcription factors like GATA and FOG1 affect the size of the platelets size [61].
The impact of rs6141 of THPO gene on protein structure was evaluated by protein dynamics studies using GROMACS. It showed that the wild type structure of THPO was stable around 2.0 nm (3.5 ns) with respect to its crystal structure. On the other hand mutated (R38C) structure showed a sudden jump to higher RMSD value (2.5 nm) at 4.0 ns and maintained a constant higher RMSD value during the course of its simulation. Mutant protein encoded by THPO (rs6141) gene also showed a reduced compactness in structure in comparison with the wild type protein. This suggested that a minor deviation between the wild and mutated RMSD values affects the original protein structure.
ARHGEF3, also known as XPLN is an exchange factor found in platelets, leukemics, and neuronal tissues [62]. It was first identified as RhoGEF (Rho guanine nucleotide exchange factor) for Rho GTPases through an expressed sequence tag database search, using the diffused B-cell lymphoma (Dbl) homology (DH) domain query in the BLASTN system [62]. Skeletal muscles and the brain have the highest levels of ARHGEF3 protein expression, followed by the heart, kidneys, platelets, and macrophages [63]. It plays a non-canonical role by inhibiting mTORC2 kinase activity through Akt signaling. [63, 64]. It is also involved in various primary cellular functions including cell adhesion, motility, polarity, growth, cell diferentiation and cytoskeleton rearrangements [63, 65].
GWAS studies have identified newer roles of ARHGEF3 gene in modulating bone mineral density (BMD), platelet differentiation and Hirschsprung disease [22, 66]. Another GWAS carried out to evaluate the association of significant variants with platelet traits reported the association of rs1354034 with MPV, in association with other genes including WDR66, TAOK1, and Phospatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma [22]. A meta-analysis including the results of various GWAS studies on 66, 867 European individuals also demonstarted that rs1354034 located at 3p14.3 is assocaited significantly with PLT count and MPV [50]. Zou et al (2017), found that this SNP is present in the regulatory region (non-coding region upstream of the transcription start site) of ARHGEF3 gene and proposed that it may influence the binding of certain trasnscription factors like RUNX1, MEIS1, GATA2, GATA1, and FLI1 duirng MK maturation (Fig. 15). However, it is not clear if this SNP is directly involved in influencing the binding sites of these transcription factors [66]. The C allele of rs1354034 has been associated with lower ARHGEF3 mRNA expression, higher PLT count and lower MPV in humans (Zou et al., 2017). In a very recent report researchers tried to investigate the genetic overlap of platelet parameters with an endophenotype of Parkinson’s Disease. They found that various genes including ARHGEF3 are associated with MPV as well as age of onset and Parkinson’s Disease suceptibility [67].
In the current study evaluating the association of rs1354034 (ARHGEF3) with IS and its subtypes, we found a significant association of CC genotype and C allele with the disease which was confirmed by MLR analysis showing an independent association of CC genotype and C allele with the disease. As far as association of this variant with IS subtypes is concerned, CC genotype and C allele were found to be associated significantly with LAA, cardioembolism, small artery occlusion and stroke of undetermined etiology.
We also evaluated the association of variant genotypes of ARHGEF3 gene with MPV, Clot rate and PLT count. The variants CC and TC genotypes showed a significant association with elevated MPV, higher clot rate and reduced PLT count in comparison with TT genotype. This association was also confimred by MLR. A GWAS reported that the same SNP (rs1354034) is associated in trans with expression of vWF, which is an important factor in blood coagulation pathway in humans [68].
Expression analysis revealed that patients bearing CC genotype of ARHGEF3 gene showed highest expression followed by TC genotype in comparison with the TT genotype in platelets.
Since this variant is an intronic variant its impact on ARHGEF3 mRNA was evaluated using UNAFold. This analysis revealed that the mRNA encoded by CC genotype of ARHGEF3 gene (rs1354034) leads to qualitatively greater stability with respect to the free energy associated with the secondary structure as compared to the mRNA encoded by normal genotype.
This SNP present upstream to the ARHGEF3 gene has been associated significantly with higher expression of ARHGEF3 during MK maturation both in murines and humans [66, 69, 70]. Based on the previous studies it has been reported that ARHGEF3 is invloved in platelet shape change and function. It has also been demonstrated that ARHGEF3 might be a missing link between ADP mediated platelet shape change and activation via P2Y1 and P2Y2 receptors [66]. The proposed mechanism by which CC genotype (rs1354034) might lead to the higher expression of ARHGEF3 gene activating the MK maturation in bone marrow producing enlarged platelets has been depicted in Fig. 16. Studies on mice lacking P2Y1 receptor do not show shape change of platelets in response to ADP suggesting that the ADP signaling is associated with shape change mechanisms in these blood cells [71]. This observation was further aided by another study that reported platelet shape change occurs through Rho signaling and actin reorganization [70, 72].
As mentioned previously THPO is a glycoprotein produced primarily in the liver that stimulates the formation of megakaryocytes and platelets. THPO protein was found to interact with 10 other proteins mainly involved in platelet activation (c-MPL, IL3), platelet aggregation (IL3), erythropoiesis (EPO, STAT5B), megakaryocyte development (c-MPL, JAK2, STAT3), cytoskeleton organization (CSF3), cell survival and proliferation (KITLG, STAT5A) (Fig. 17) [73–75]. For the production of platelets MKs undergo a series of remodeling events that result in thousands of platelets being released from a single cell [76]. All the proteins found to be interact with THPO protein are known to regulate the platelet formation and functions [77–79]. The interaction of THPO protein with other proteins suggested that there might be potential alternate mechanisms that could affect platelet production, morphology and function. The THPO along with other interacting proteins might be explored as significant biomarker affecting platelet parameters and functions and thereby a potential therapeutic target.
ARHGEF3 activates two members of the Rho family GTPases, RHOA and RHOB, which are involved in osteoblast maintenance [70]. Various other cellular processes including cytoskeleton reorganization are activated and inactivated by Rho-like GTPases as discussed previously [80, 81]. By catalyzing the release of bound GDP, guanine nucleotide exchange factors (GEFs) accelerate Rho GTPase activity. ARHGEF3 inhibits mTORC2 kinase activity, primarily for Akt, by binding the mTORC2 complex. (Arthur et al., 2002; Rossman et al., 2005). ARHGEF3 protein has been found to interact with five other proteins involved in cytoskeleton organization (RHOA, RHOC), cell adhesion, migration (RHOA, RHOC, AHOB, and CTNNB1), apoptosis (RHOB) and cholesterol homeostasis (RNF145) (Fig. 18). Many physiological and pathological functions of platelets are mediated by Rho GTPase proteins [80]. Actin cytoskeleton regulation is one of the main functions of Rho GTPases, although they also participate in several other biochemical pathways [82]. When platelets interact with vWF and collagen via the cell-surface receptors GpIb-IX-V and GPVI, respectively, a dramatic change in shape occurs due to the reorganization of the actin cytoskeleton. When platelet morphology is altered, more surface area is available for interactions with the ECM and other cells [83, 84]. Initial shape changes include discoid loss, sphering, and filopodia extension. The interaction of ARHGEF3 and THPO with other proteins significantly involved in various platelet parameters and functions suggests that the genotype-phenotype correlation should not be based on one protein but rather than complete network should be analysed to explore their role as biomarkers or therapeutic targets.