In a recent study published in Thrombosis Research, researchers determined plasma signatures of platelet-related or derived proteins in isolated acute pulmonary embolism (iPE) and PE associated with deep vein thrombosis (DVT-PE), in compared with isolated DVT (iDVT).
Study: Subtype-specific plasma signatures of platelet-related proteins released in acute pulmonary embolism. Image credit: Victor Josan/Shutterstock
background
The two primary subtypes of PE are iPE and DVT-PE, and studies have shown that the plasma protein profiles for the two conditions differ. Platelets act as cellular linkers facilitating inflammatory crosstalk between immune and endothelial cells under high arterial shear stress. This process is a direct receptor-mediated interaction involving proinflammatory molecules and extracellular vesicles (EVs). However, local inflammatory responses in the endothelium at low shear stress activate platelets to initiate the development of venous thromboembolism (VTE) and its subtypes, including PE and DVT. The mechanisms governing this process are less well known.
Experimental data suggest that platelets contribute to thromboinflammation of the venous system by causing inflammatory and coagulation processes. For example, using the murine model of VTE, researchers have shown that platelets interact with endothelial cells by exposing von Willebrand factor (VWF) and forming conjugates with leukocytes via glycoprotein (GP) Ibα, as well as triggering endothelial recruitment and leukocyte-dependent coagulation. .
Human experiments have shown differential properties of platelet activation and reactivity in acute VTE. For example, platelets from patients with acute VTE showed more exocytosis of dense granules and lysosomes. It was accompanied by higher plasma levels of thromboxane B2 but less platelet-dependent thrombin generation than patients with excluded VTE, regardless of aspirin therapy.
Mass spectrometry (MS) studies have revealed over 3700 proteins in resting, inhibited and activated human platelets. Advanced enzyme-linked immunosorbent assays (ELISA) with MS could allow the qualitative assessment of platelet-released proteins in plasma and isolated platelets. However, a more detailed analysis of platelet-associated plasma proteins in large VTE cohorts is lacking.
About the study
In the present multicenter prospective cohort study, researchers profiled plasma collected from 541 VTE patients using machine learning-based analysis. The aim was to identify plasma protein signatures for putative platelet release, specific for iPE and DVT-PE. These patients had acute VTE at enrollment as diagnosed by imaging. While there were 99 iPE patients, 282 were DVT-PE patients, and the team compared their data with 160 iDVT patients. The team used whole-leg color Doppler ultrasound and computed tomography (CT) pulmonary angiography for DVT and PE diagnoses. Board-certified angiologists and radiologists adjudicated and validated all study diagnoses.
They collected study samples as part of the Venous Thromboembolism Genotyping and Molecular Phenotyping (GMP-VTE) project conducted in Germany. The researchers used proximity extension assay (PEA) technology to profile abundant and low plasma proteins from samples stored at -80°C. PEA obtained normalized expression (NPX) values for all tested plasma proteins incorporating oligonucleotide-labeled antibodies and quantitative real-time polymerase chain reaction (PCR) amplification.
The assay panel consisted of 444 proteins identified from five databases [e.g., Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) databases]. After comparing, the team eventually identified 135 platelet-related proteins in the five PEA panels for further analysis.
Results of the study
The study cohort demonstrated a higher prevalence of hypertension, diabetes, and chronic inflammatory, atherosclerotic, and cardiovascular diseases in iPE and DVT-PE compared with iDVT. Higher levels of C-reactive protein (CRP), troponin I, and N-terminal prohormone (NT) B-type natriuretic peptide (NT-proBNP) similarly reflected greater cardiovascular burden in PE subtypes.
Drug prescribing patterns varied accordingly. For example, antiplatelet therapy, namely acetylsalicylic acid (ASA) and clopidogrel, were overrepresented among PE patients. One explanation is that prophylactic antiplatelet medication is prescribed for suspected myocardial infarction in patients with acute PE. It is also possible that there is variable platelet activity between patients with PE and iDVT because the proportion of antiplatelet agents is higher in the PE groups.
Machine learning analysis of 135 platelet proteins extracted by the minimum absolute shrinkage and selection operator (LASSO) selected 24% and 22% for iPE and DVT-PE, respectively, which reflected different profiles of proteins compared to iDVT. In particular, the 135 platelet proteins demonstrated a good association with six markers of platelet activation, supporting their probable platelet origin in the plasma of patients with acute PE compared to patients with iDVT, analyzed using the PEA panels. In contrast to iPE, stromal cell-derived factor 1alpha (SDF-1α) was highly expressed in DVT-PE than in iDVT patients, indicating a potentially different role in vascular inflammation and atherogenesis
In iPE, protein-protein interaction (PPI) network analysis resulted in four clusters of up to six functionally interacting proteins based on 22 platelet-related proteins specifically expressed compared to iDVT. The main cluster was related to adhesion, pattern recognition and immune receptor signaling. These covered the c-Src Src family kinases (SFKs), which transduce ligand signaling through platelet receptors associated with the tyrosine-based activation motif (ITAM) of immunoreceptors (eg, glycoprotein VI). [GPVI]).
In relation to iPE, DVT-PE presented a group of nine directly interacting plasma proteins related to platelets involved in tissue remodeling and leukocyte trafficking. Tissue inhibitors of matrix metalloproteinases 1 (TIMP1) and TIMP4, prominent effectors of tissue remodeling, were more highly expressed in DVT-PE than in iDVT and secreted from platelet α-granules.
The authors noted that higher plasma levels of tissue inhibitors of TIMP1 and TIMP4 were associated with type 2 diabetes, hypertension, and myocardial infarction, which is in line with the higher prevalence of major cardiovascular events in the DVT-PE than in iDVT.
Conclusions
The results of the current study revealed that both PE subtypes exhibited specific profiles of platelet-associated plasma proteins. For example, the study differentiated a higher expression of P-selectin in the plasma of patients with DVT-PE compared to iDVT, suggesting a relationship with DVT disease severity. Interestingly, thrombin peak height and spontaneous platelet aggregation in platelet-rich plasma were negatively related in the iPE compared with the iDVT phenotype. These results suggested that lower platelet reactivity in vitro might be associated with higher platelet activation in vivo during the acute phase of PE compared with iDVT. More importantly, these results suggested that although PE subtypes share some common patterns, they also exhibit different patterns of platelet activation.
This study did not address the quantification and characterization of EV in plasma of VTE phenotypes. In cancer patients, VTE could be related to increased plasma levels of microparticles. However, in patients without cancer, only a significant increase in platelet-derived microparticles was observed with recurrent VTE compared with healthy blood donors. Future studies should clarify the distribution of EV in the different VTE phenotypes. Further studies are also needed to specify the impact of different cell types on the release of platelet-derived proteins in PE.
In conclusion, the study data indicated that iPE and DVT-PE exhibited specific but variable plasma signatures involved in immunothrombosis and platelet-related thromboinflammatory processes compared with iDVT. Furthermore, platelet activation protein profiles appeared to differ between PE subtypes, with a preponderance of proteins secreted in DVT-PE compared with proteins more likely to be released into plasma by EVs in iPE. In general, platelets contribute to regulating different levels of plasma proteins in the acute phase of PE that differ between PE subtypes.