Karen P. Fong

Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear‐resistance of platelet aggregates

Summary.  Background: Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1–10 mL of blood and are poorly suited for murine whole blood experiments. Objective: To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 μL of whole blood and controlled flow exposure. Methods: Microfluidic methods were used for patterning acid‐soluble collagen in 100 μm × 100 μm patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti‐mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin α2 subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a ‘shear step‐up’ to 8000 s−1. Results: Wild‐type murine platelets adhered and aggregated on collagen in a biphasic shear‐dependent manner with increased deposition from 100 to 400 s−1, but decreased deposition at 1000 s−1. Adhesion to patterned collagen was severely diminished for platelets lacking a functional α2β1 integrin. Those integrin α2‐deficient platelets that did adhere were removed from the surface when challenged to shear step‐up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step‐up. Conclusions: PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombin‐dependent pathways such as fibrin formation.

Deciphering the human platelet sheddome

Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function.

RGS/Gi2α interactions modulate platelet accumulation and thrombus formation at sites of vascular injury

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I(2) (PGI(2)), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in G(i2α) that blocks RGS/G(i2) interactions to examine the consequences of lifting constraints on G(i2)-dependent signaling without altering receptor:effector coupling. The results show that the G(i2α)(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca(++)](i), Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Molecular priming of Lyn by GPVI enables an immune receptor to adopt a hemostatic role

The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates.

Disruption of SEMA4D ameliorates platelet hypersensitivity in dyslipidemia and confers protection against the development of atherosclerosis.

Objective—In dyslipidemic states, platelets become hyperreactive, secreting molecules that promote atherosclerosis. We have shown that the semaphorin family member, sema4D (CD100), is expressed on the surface of platelets and proposed that its role includes promoting thrombus growth by binding to nearby platelets and endothelial cells, both of which express sema4D receptors. Here we tested the hypothesis that deleting sema4D will attenuate the adverse consequences of dyslipidemia on platelets and the vessel wall. Methods and Results—Platelet function and atherosclerotic lesion formation were measured in LDLR(−/−) and sema4D(−/−)LDLR(−/−) mice after 6 months on a high-fat diet. All of the mice developed the dyslipidemia expected on this diet in the absence of functional LDL receptors. However, when compared to LDLR(−/−) mice, sema4D(−/−) LDLR(−/−) mice had reduced lipid deposition in the descending aorta, a 6-fold decrease in the frequency of arterial occlusion and a reduction to near wild-type levels in the accumulation of platelets after injury. These differences were retained ex vivo, with a marked decrease in platelet accumulation on collagen under flow and in platelet aggregation. Conclusions—These results show that loss of sema4D expression reduces the platelet hyperactivity otherwise found in dyslipidemia, and confers protection against the development of atherosclerosis.

Aggregatibacter actinomycetemcomitans leukotoxin requires β‐sheets 1 and 2 of the human CD11a β‐propeller for cytotoxicity

Aggregatibacter actinomycetemcomitans leukotoxin (Ltx) is a repeats‐in‐toxin (RTX) cytolysin that kills human leukocyte function‐associated antigen‐1 (LFA‐1; αL/β2)‐bearing cells. In order to determine whether the αL portion of the heterodimer is involved in Ltx recognition, we transfected human, mouse and bovine αL cDNAs into J‐β2.7, an αL‐deficient cell line, and looked for restoration of Ltx susceptibility. Cells expressing either bovine or human αL in conjunction with human β2 were efficiently killed by Ltx, an indication that bovine αL could substitute for its human counterpart in critical regions used by Ltx for attachment to LFA‐1. On the other hand, cells expressing murine αL and human β2 were not susceptible to the lethal effects of Ltx indicating that the toxin recognition sites are not present in the corresponding mouse sequence. To further identify the region(s) of αL recognized by Ltx, we constructed and evaluated a panel of chimeric human/murine αL genes in J‐β2.7 cells. Analysis of the αL mutant panel showed that the presence of human N‐terminal 128 amino acids on a mouse CD11a background, a region that includes β‐sheets 1 and 2 of the β‐propeller of the human αL chain, was sufficient for Ltx cytolysis.

Diminished contact-dependent reinforcement of Syk activation underlies impaired thrombus growth in mice lacking Semaphorin 4D.

We recently reported that Semaphorin 4D (Sema4D) and its receptors are expressed on the platelet surface and showed that Sema4D((-/-)) mice have a selective defect in collagen-induced platelet aggregation and an impaired vascular injury response. Here we investigated the mechanisms involved, tested the role of platelet-platelet contacts in Sema4D-mediated events, and examined the relationship between Sema4D-dependent signaling and integrin α(IIb)β(3) outside-in signaling. The results show that spleen tyrosine kinase (Syk) activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRγ complex, is greatly reduced in Sema4D((-/-)) platelets and can be restored by adding soluble Sema4D. Earlier events, including FcRγ phosphorylation, occur normally; later events are impaired. In contrast, when engagement of α(IIb)β(3) was blocked, Sema4D((-/-)) and control platelets were indistinguishable in assays of Syk activation, adhesion, spreading on collagen, and activation of α(IIb)β(3). Finally, we found that, unlike the Sema4D knockout, α(IIb)β(3) blockade inhibited FcRγ phosphorylation and that stimulating aggregation with Mn(2+) failed to normalize Syk activation in the absence of Sema4D. Collectively, these results show that α(IIb)β(3) and Sema4D jointly promote collagen responses by amplifying Syk activation, partly by forming integrin-mediated contacts that enable the binding of Sema4D to its receptors and partly through integrin outside-in signaling. These 2 processes are interdependent, but distinguishable.

Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains.

Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27-29%) and palmitolyl (C16:1 cis Δ9, 43-44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12-14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys(562) and Lys(687) are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coliα-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys(562) and Lys(687) with Arg blocks acylation, resulting in a >75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these post-translational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity.

Dynamic Spreading of Platelets on Collagen in Microchannels

Platelets come contact collagen exposed on the subendothelial matrix at sites of vascular injury that triggers their activation and the formation of a hemostatic plug. Glycoprotein VI and integrin α2 β1 are major collagen receptors on the platelet surface. Although the spreading of platelets on the collagen is important to function, it has been difficult to study the dynamics of spreading over a relevant time scale. Here we focus on the early stages of murine platelet spreading on collagen and/or fibrinogen under both static conditions and flow and then probe their dynamics by quantitative visualization using real-time reflection interference contrast microscopy. In this study, we found under static conditions the spreading area of platelets on collagen initially increased quickly by following the power law A ∼ t0.7 before slowing. Interestingly, we observed in microchannels under flow that single platelets as well as aggregates that were spread on the collagen contracted over time under shear. This contraction was not observed under static conditions in our system. This effect might help to maintain the hemostatic plug under the shear force. Future studies will be aimed at investigating the spatial-temporal cytoskeletal dynamics of platelets and the functions of collagen receptors on the platelets surface using platelets derived from genetically engineered mice.Copyright