Monash researchers have developed a drug that can be potentially given as a preventative against heart attack.
The drug – which has been studied in human cells and animal models – literally blocks the minute changes in blood flow that preempts a heart attack and acts on the platelets preventing the platelet-triggered clot before it can kill or cause damage.
Importantly, the drug may have a role in preventing the clotting that is the hallmark of COVID-19.
One third of all deaths globally – 18 million a year – are caused by cardiovascular disease, largely heart attack or stroke, both of which are triggered by clots blocking the vessels in the brain or heart.
While drugs like aspirin, given at the time of the attack, can prevent further clots forming, they only work in 25 per cent of cases, and these drugs can cause serious side effects from bleeding.
According to the lead scientist on the paper, published in the prestigious journal, Science Translational Medicine, Associate Professor Justin Hamilton, from the Monash University Australian Centre of Blood Diseases, “there has been no new drugs to treat, let alone prevent, heart attack or stroke in more than 15 years,” he said.
Associate Professor Hamilton said the researchers stumbled across the potential drug by accident. They were looking at changes within platelets that occur around the time of what is called a pathological setting, ie a heart attack or stroke.
They found an enzyme of interest, isolated the gene responsible and developed a mouse that was missing just that gene.
The mice – to their surprise – were completely protected against heart attack. But why this enzyme provided protection remained a mystery for two years. “It drove us mad,” Associate Professor Hamilton said.
The researchers used electron microscopy to cut ultrathin “slices” of the platelets from these mice to see what was going on. What they saw was a slightly modified membrane, which appears to prevent these platelets from attaching to each other or to blood vessel walls, the minute that there is a change in blood flow.
“It is this blood flow perturbation which is a hallmark and predictor of a heart attack,” Associate Professor Hamilton said.
“This enzyme allows the platelets to respond to this blood flow change and to “gear up” their capacity to clot, causing an attack.”
Once the researchers were aware of the importance of the enzyme, they developed a drug that could shut this process down, in animal models and in laboratory models using human blood.
This drug has the potential to be given to patients at risk of heart attack and stroke, to prevent blood clots forming when there is a risk of attack.
The next step is to develop a more suitable drug candidate that could be taken into a clinical trial, according to Associate Professor Hamilton. Initially he is hoping to test the drug on patients who have a higher risk of cardiovascular disease, such as those with diabetes.
These same clots – targeted by the Monash drug – have recently been linked to COVID-19 as a key cause of death from the disease.
Associate Professor Hamilton said that, while it is early days, “the possibility of using our newly developed anti-thrombotic to improve the treatment of COVID-19 patients is an appealing idea we would like to explore.”
The phosphoinositide 3‐kinases (PI3Ks) are a family of lipid kinases that catalyse the phosphorylation of the 3′ hydroxyl group of the inositol ring of phosphoinositides to produce the 3‐phosphorylated phosphoinositides – important second messengers for a wide range of processes in cells [1, 2].
There are eight mammalian PI3K isoforms, divided into three classes based on structural and functional similarities: four class I, three class II, and one class III PI3K. The class I PI3Ks are comfortably the most widely studied and have well‐defined roles in range of cells, including platelets .
More recently, mouse genetic models have been used to uncover the importance of the sole class III PI3K, Vps34 [4, 5], and one of the class II PI3Ks, PI3KC2α [6-8], in platelet function.
PI3KC2α in particular regulates the thrombotic function of mouse platelets by a unique and as‐yet‐undefined mechanism that may involve the structure of the cell membrane.
Specifically, we used an inducible shRNA‐based approach to deplete PI3KC2α protein in the platelets of adult mice to show that PI3KC2α regulates the structure of the platelet internal membrane system (the open canalicular system; OCS) [6, 7].
Here, transmission electron microscopy (TEM) revealed that PI3KC2α‐deficient platelets exhibit dilatations of the channels of the OCS. Perhaps surprisingly, platelets from PI3KC2α‐deficient mice displayed normal function in all in vitro function tests performed (aggregation, granule secretion, integrin activation, and adhesion and spreading on activating surfaces), yet have impaired platelet thrombotic function in both in vitro and in vivo models [6, 7].
A subsequent study confirmed this role for PI3KC2α in platelet structure and function in a distinct mouse model involving heterozygosity of a kinase‐inactivating point mutation in the PI3KC2α active site . These intriguing findings suggest PI3KC2α links regulation of the platelet internal membrane structure to the cell’s prothrombotic function. However, the mechanism by which this occurs remains unknown.
One potential mechanism by which alterations in the platelet membrane structure might impair platelet function is provided by the observation that PI3KC2α‐deficient platelets exhibit reduced cytoskeletal co‐localization of key proteins that link the cell membrane with the cytoskeleton, most notably spectrin and myosin .
If the reduction in these proteins is sufficient to impair communication between the membrane and cytoskeleton, one prediction is that this may lead to impaired filopodia formation in activated platelets.
Yet how such an impact would result in the observed selective impairment of platelet function in the setting of thrombosis – and not a global impairment of platelet function in, for example, standard assays of aggregation, granule secretion, or platelet spreading , remains unclear.
Given this uncertainty regarding the mechanism by which PI3KC2α regulates platelet membrane structure and function, we have further examined the structural changes to the platelet membrane induced by PI3KC2α‐deficiency and have investigated how these structural changes affect the function of the platelet membrane.
Here, we use focused ion beam‐scanning electron microscopy (FIB‐SEM) , as well as traditional SEM, to perform a detailed, three‐dimensional ultrastructural analysis of platelets from PI3KC2α‐deficient mice.
This analysis indicates PI3KC2α‐deficient platelets exhibit specific changes to the structure of the OCS. These changes occur without major alterations in the lipid composition of these platelets. Surprisingly, the structural changes to the platelet OCS membrane have no impact on the formation of filopodia by platelets activated in suspension, yet markedly impacts on the ability of these platelets to form thrombi in a blood flow environment involving a prominent shear gradient that produces membrane tether formation [10-12].
Together, these findings indicate that PI3KC2α modulates the structure of the platelet internal membrane (OCS) independently of changes to membrane composition. These structural changes to the OCS appear to compromise platelet membrane function specifically in the setting of thrombus formation. If this regulation of membrane structure by PI3KC2α is acute, these studies reveal the potential for a novel approach toward thrombosis‐specific anti‐platelet therapy.
PI3KC2α regulates platelet membrane structure
We have previously used two‐dimensional TEM imaging to demonstrate that PI3KC2α‐deficiency results in a dilation of the channels of the OCS within mouse platelets .
In order to examine this and any other potential ultrastructural change caused by PI3KC2α‐deficiency in more detail, we used FIB‐SEM to create whole‐cell, three‐dimensional reconstructions of PI3KC2α‐deficient mouse platelets.
These reconstructions allow for visualization and quantitation of various intracellular compartments, which in this case comprised the OCS and plasma membrane, as well as alpha and dense granules.
In agreement with our previous two‐dimensional findings, we observed dilation of the OCS in platelets from PI3KC2α‐deficient mice when compared with platelets from littermate controls (Fig. 1A).
Areas of OCS dilation in PI3KC2α‐deficient platelets were more evident at certain points in a given cell (Fig. 1A, red arrows) and the total volume of OCS in platelets isolated from PI3KC2α‐deficient mice was 48% greater than in platelets from littermate control mice (3.6 ± 0.5 vs 5.4 ± 0.6% of total cell volume for control and PI3KC2α‐deficient cells, respectively; Fig. 1B).
Despite this OCS dilation, no obvious differences were observed in the spatial distribution of the OCS throughout the cell (Fig. 1A). In contrast to the OCS, the total volume of either alpha or dense granules was not different between control and PI3KC2α‐deficient platelets (Fig. 1C).
Given the OCS dilatation observed here and previously [6, 7], we next examined the external aspect of the OCS – its openings at the plasma membrane – via SEM (Fig. 2A). There were similar numbers of OCS openings in the visible plasma membrane of cells examined in these studies (Fig. 2B).
However, in a near‐identical finding to the OCS measurements inside platelets, the diameter of OCS openings at the plasma membrane were significantly increased by 46% in platelets from PI3KC2α‐deficient mice versus littermate controls (37 ± 4 vs 54 ± 3 nm, respectively; Fig. 2C).
PI3KC2α does not regulate platelet membrane lipid composition
One of the most important contributors to membrane structure is the lipid content of the membrane. Therefore, we next examined whether the observed structural changes to the OCS were caused by or associated with alterations in the lipid profile of the platelet.
We performed a detailed analysis of 294 lipids across the 22 most abundant lipid classes and subclasses, using liquid chromatography electrospray ionization‐tandem mass spectrometry.
This analysis revealed an unchanged lipid profile in PI3KC2α‐deficient when compared with littermate control platelets (Fig. 3), with no significant differences in molar abundance observed in any of the lipid classes analysed.
PI3KC2α does not regulate filopodia formation in platelets
We next examined whether the structural alteration in the platelet internal membrane impacts on platelet membrane function by imaging filopodia formation in platelets activated in suspension or in the presence of shear forces.
First, isolated platelets were stimulated with ADP (in the presence of eptifibatide to prevent aggregation), then fixed and imaged by SEM (Fig. 4A). No differences were detected in either the number or length of filopodia between platelets isolated from PI3KC2α‐deficient or littermate control mice (Fig. 4B,C).
In addition, filopodia formed by platelets flowed over a monolayer of activated platelets were also examined. Again, no differences in tether length or stability (total time of attachment) were observed (Fig. 4D,E).
PI3KC2α regulates platelet adhesion and thrombus formation
Given the impaired arterial thrombus formation previously observed in PI3KC2α‐deficient mice, we examined platelet function in a microfluidic whole blood flow assay known to be dependent on membrane tether formation [10-12]. Mouse blood was perfused through a von Willebrand factor‐coated microfluidic channel incorporating a shear force gradient‐inducing stenosis [10-12]. Platelet aggregation occurred downstream of the stenosis and the size of these platelet aggregates was quantified, using the epifluorescent signal of the GFP‐positive platelets (Fig. 5). The extent of platelet deposition was significantly lower in blood from PI3KC2α‐deficient mice (838 ± 314 μm2) versus that in blood from control mice (2194 ± 485 μm2) – a 62% reduction (Fig. 5).
Together, these findings suggest loss of PI3KC2α leads to a specific structural change in the platelet membrane that occurs independently of membrane lipid composition. This membrane modification is sufficient to selectively impact upon platelet function in the setting of thromboses formed downstream of a shear gradient‐inducing stenosis and dependent on membrane tethering.
We performed an analysis of the structural and functional consequences of the loss of PI3KC2α in mouse platelets in order to gain insights into the mechanism by which PI3KC2α deficiency leads to the previously observed anti‐thrombotic phenotype.
We showed that PI3KC2α deficiency leads to an ultrastructural change in platelets that is limited to the internal membrane (OCS) of these cells. Specifically, the OCS in platelets from PI3KC2α‐deficient mice was dilated throughout the cell, including at the plasma membrane.
This structural change in the platelet membrane occurred independently of the cell’s lipid composition. Yet inspection of platelet membrane function revealed PI3KC2α deficiency had selective effects under prothrombotic conditions: filopodia formation was unaffected, but platelet deposition and aggregation was significantly impaired in an ex vivo whole blood flow thrombosis model.
We and others have previously shown that platelets from PI3KC2α‐deficient mice exhibit an increased surface area occupied by the channels of the OCS in TEM images [6, 8]. Here, we add to those findings to show that the enlargements previously reported in 2D TEM images are reflective of a widespread and largely uniform dilation of membrane channels throughout the cell, including at the plasma membrane.
Our detailed three‐dimensional analysis of PI3KC2α‐deficient platelets revealed this structural change was specific to the OCS: its volume was significantly increased in PI3KC2α‐deficient platelets while the volumes of alpha and dense granules were unaffected, despite the occasional change in granule number or shape between individual cells.
Furthermore, the size of the openings of the OCS at the plasma membrane were significantly larger in PI3KC2α‐deficient platelets. Despite this OCS dilation throughout the cell, no gross change in the spatial distribution of the OCS across the cell was observed.
Despite these obvious changes in the structure of the cell’s membrane, it remains unclear how PI3KC2α‐deficiency leads to such changes. It has previously been shown that PI3KC2α‐deficiency reduces the basal levels of PI(3)P in platelets .
Yet, this small change in a low abundance lipid appears unlikely to directly cause the structural changes in the platelet membrane observed here. Given that the biggest predictor of any membrane structure is its lipid composition, here we expanded on these previous studies by performing a comprehensive analysis of the lipidome of both PI3KC2α‐deficient and wild‐type platelets.
This more broad‐based analysis included all of the most abundant membrane lipid species and revealed the gross lipid profile of PI3KC2α‐deficient platelets was indistinguishable to that of wild‐type platelets. These studies indicate the observed internal membrane dilatation is unlikely driven by compositional changes to the membrane.
This is, to our knowledge, the most comprehensive lipidomic analysis of the mouse platelet. On this, it is interesting to note some potential differences in the relative abundance of some lipids between mouse platelets and similar lipidomic analyses of human platelets .
For example, while phospholipids are the predominant component in both mouse and human platelet membranes, there is a notably higher abundance of sterol lipids and a relative paucity of glycerolipids in mouse platelets when compared with human . Whether these differences are important in terms of membrane function remains to be determined.
Previous studies have demonstrated an in vivo antithrombotic phenotype conferred by loss of PI3KC2α [6, 8]. Given that platelets from these PI3KC2α‐deficient mice have a structural defect that is specific to the OCS – a reserve of membrane thought to be utilized for platelet shape change and spreading during activation and aggregation  – we examined platelet membrane function in both static and blood flow environments.
We found that platelet membrane function is affected specifically in the setting of thrombus formation. Here, filopodia production in suspension‐activated platelets or under flow were unaffected by loss of PI3KC2α, yet in a microfluidic assay incorporating a high shear gradient‐inducing stenosis, PI3KC2α‐deficiency led to a marked reduction in platelet deposition.
We utilized this microfluidic blood flow assay because of its incorporation of a pronounced stenosis that has been shown to cause a substantial shear gradient (from very high at the point of stenotic narrowing, to very low immediately downstream of the stenosis) [10-12].
Importantly, in vivo studies of platelet behaviour following exposure to such a shear gradient environment (via a vascular stenosis) have shown that initial platelet deposition occurs largely independently of cellular activation by soluble agonists .
Indeed, such shear gradients appear to drive initial platelet deposition via the formation of membrane tethers [10-12]. As a result, the significant reduction in platelet deposition in this system using blood from PI3KC2α mice may suggest that PI3KC2α is important for the earliest stages of platelet adhesion, where membrane tethers, presumably derived from the OCS, are pulled from platelets without detectable cell activation via an interaction between von Willebrand factor and the platelet adhesion receptor GPIbα .
This hypothesis fits our previous observations in which PI3KC2α‐deficient platelet adhesion and thrombus formation is differentially affected under conditions in which platelet activation is substantial, that is, on a collagen‐coated surface upon which marked platelet activation occurs .
Alternatively, PI3KC2α may be involved in the subsequent propagation of platelet aggregates, since incoming platelets that tether to the surface of forming thrombi are discoid and demonstrate low levels of activation markers and minimal calcium flux, in sharp contrast to the initial core of the thrombus where platelets are generally fully activated [12, 18].
The lack of effect of PI3KC2α deficiency on platelet filopodia production in response to cell activation may suggest that the involvement of PI3KC2α is most prevalent where changes in shear stress sustain and drive thrombus propagation, potentially in the absence of conspicuous agonist‐induced activation. The surprising lack of effect of PI3KC2α deficiency in standard assays of agonist‐induced in vitro platelet function [6, 8] supports this hypothesis.
The intriguing link between the PI3KC2α‐dependent modulation of platelet internal membrane structure and impaired platelet function specifically in the setting of blood flow suggests targeting PI3KC2α and/or the platelet internal membrane system may have utility as an anti‐thrombotic strategy. However, it remains unknown whether this function of PI3KC2α translates to human platelets.
In addition, the relevance of changes in platelet membrane structure to human platelet function are completely unexplored. There are a number of clinical syndromes in which abnormalities in the OCS are observed, including some with either bleeding or thrombotic consequences [19-21].
Yet platelets from patients with these conditions demonstrate a number of structural abnormalities in addition to OCS changes, making it difficult to determine how much, if any, contribution the OCS defect has to the clinical phenotype.
The development of PI3KC2α inhibitors would be a useful step forward in determining the role of this enzyme in human platelets and in addressing any validity of this target as an antithrombotic approach.
If PI3KC2α function is conserved in human platelets and acute inhibition of this enzyme is able to reproduce the phenotype observed in mouse platelets, this opens the possibility of manipulating the platelet membrane structure via PI3KC2α as a novel, thrombosis‐specific, antiplatelet strategy.
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More information: Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation, Science Translational Medicine (2020). DOI: 10.1126/scitranslmed.aar8430