caClot, or not? Reviewing the reciprocal regulation between lipids and blood clotting

Abstract

Both Hyperlipidemia and thrombosis contribute to the risks of atherosclerotic cardiovascular diseases, which are the leading cause of death and reduced quality of life in survivors worldwide. The accumulation of lipid-rich plaques on arterial walls eventually leads to the rupture or erosion of vulnerable lesions, triggering excessive blood clotting and leading to adverse thrombotic events. Lipoproteins are highly dynamic particles that circulate in blood and carry insoluble lipids and are associated with proteins, many of which are involved in blood clotting. A growing body of evidence suggests a reciprocal regulatory relationship between blood clotting and lipid metabolism. In this review article, we summarize the observations that lipoproteins and lipids impact the hemostatic system, and the clotting-related proteins influence lipid metabolism. We also highlight the gaps that need to be filled in this area of research.

Graphical Abstract

1. Introduction

Atherosclerotic cardiovascular diseases (ASCVDs), including myocardial infarction (MI) and ischemic stroke, are the leading causes of death and reduced quality of life in survivors worldwide^1,2. These diseases share common features in their pathological progression from hyperlipidemia to lipid-rich plaques building up on arterial walls to the rupture or erosion of vulnerable lesions. Eventually, this triggers excessive clotting, leading to adverse thrombotic events that block or reduce blood flow and cause tissue ischemia. Growing evidence suggests that these common features are not happening as a coincidence, but rather have mechanistic links between hyperlipidemia and thrombosis.

Hemostasis is the mechanism that forms blood clots to stop bleeding. Blood clotting follows four steps: vasoconstriction, platelet plug formation, coagulation, and fibrinolysis. Thrombosis occurs when excessive blood clots extend and block blood flow. Lipoproteins are highly dynamic particles that circulate in blood and carry both insoluble lipids and are associated with proteins, many of which are involved in blood clotting. In this article, we will review insights into how the blood clotting system intertwines with lipoproteins based on evidence from both clinical observation and mechanistic studies, with perspectives toward future research directions.

2. Overview of hemostatic system and lipoproteins

Primary hemostasis.

Upon vascular injury, activated endothelial cells secrete von Willebrand factor (VWF) onto the surface of lesioned endothelium and the subendothelial extracellular matrix^3,4. Platelets adhere to VWF at the injury site, prior to becoming activated and forming aggregates that lead to platelet plug formation.

Secondary hemostasis.

These platelet plugs are loose and require stabilization by crosslinked fibrin clots, which are generated through coagulation. Under physiological conditions, following vascular injury, extravascular tissue factor (TF) comes in contact with blood and forms a one-to-one complex with the serine protease activated factor VII (FVIIa)⁵, initiating the extrinsic coagulation pathway, also called the TF pathway (Figure 1). This TF-FVIIa complex then activates factor X (FX) to the serine protease FX (FXa)⁶. The TF-FVIIa complex also feeds into the intrinsic pathway by activating factor IX (FIX) to activated FIX (FIXa), which is a part of the intrinsic pathway. FIXa forms an enzymatic complex with its cofactor factor VIIIa (FVIIIa) upon activation by thrombin. The FIXa-FVIIIa complex rapidly converts FX to FXa⁷. The alternative intrinsic pathway can be initiated with the sequential activation of the serine proteases factor XII (FXIIa), factor XI (FXIa) and FIXa. FXIIa activates FXI to FXIa and prekallikrein to kallikrein, which subsequently activates more factor XII, generating more FXa⁸. Subsequently, FXa forms an one-to-one complex with factor Va (FVa) in the presence of calcium and phospholipids⁹. This complex, also called prothrombinase, converts prothrombin (FII) to thrombin (FIIa)¹⁰. Upon activation of FX to FXa, tissue factor pathway inhibitor (TFPI) strongly inhibits extrinsic pathway^11,12, without inhibition on the intrinsic pathway. Therefore, the intrinsic pathway plays a significant role in the amplification of the coagulation cascade. Thrombin is the enzyme that converts fibrinogen (FI) to fibrin, which is then cross-linked by a transglutaminase, factor XIIIa (FXIIIa), eventually generating stable insoluble fibrin clots¹³. Thrombin also amplifies its own production, through feedback activation of FVa, FVIIIa and FXIa. The anti-coagulation system naturally exists and is an important part of the hemostatic system to check and balance the coagulation cascade. The major players in the anti-coagulation system include the protein C, protein S, antithrombin, and the above mentioned TFPI.

Figure 1. Summary of the hemostatic system.

Upon vascular injury, endothelial cells secrete VWF, which platelets bind to, forming long strings and aggregates (platelet plugs). The extrinsic and intrinsic coagulation pathways work in concert to activate FX to FXa. FXa activates prothrombin to thrombin, which cleaves fibrinopeptides from the N-terminus of fibrinogen to form fibrin. Fibrin polymerizes and is cross-linked by activated FXIIIa. The polymerized fibrin stabilizes the blood clot.

Fibrinolysis.

After the cross-linked fibrin stabilized the clot, fibrinolysis gradually removes the clot, allowing blood to re-flow. Tissue type plasminogen activator (tPA) initiates fibrinolysis by binding to fibrin, where it converts the zymogen plasminogen to the active enzyme plasmin. Plasmin cleaves the insoluble crosslinked fibrin to soluble fibrin degradation products^14,15. As a positive feedback loop, plasmin also activates urokinase plasminogen activator (uPA) to produce more plasmin and amplify fibrinolysis¹⁵. PAI-1, a serine protease inhibitor (serpin), inactivates tPA or uPA by forming tPA-PAI-1 or uPA-PAI-1 complex, thereby inhibiting fibrinolysis¹⁵.

Lipoproteins.

Abundant lipid-bound proteins are carried on lipoproteins in circulation, including many involved in blood clotting. Lipoproteins are primarily known for their functions of carrying various insoluble lipids travelling through bloodstream. Regardless of the heterogeneity of lipoproteins, all contain a core with cholesterol-esters and triglycerides, surrounded by free cholesterol, phospholipids, and apolipoproteins. All apoB-containing lipoproteins are atherogenic, including chylomicron, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and Lipoprotein(a) (Lp(a)). In contrast, apoA1-containing high-density lipoprotein (HDL) are athero-protective.

3. Connections between lipoproteins and hemostatic system

3.1. Clotting-related proteins are associated with lipoproteins by proteomics

VLDL, LDL, HDL, and Lp(a) are composed of 7%, 20%, 40–55%, and 25% proteins by weight, respectively¹⁶. Proteomic analyses of isolated lipoproteins identified proteins outside of those that regulate lipid metabolism, including many proteins involved in blood clotting (Figure 2). VLDL fractions have VWF, platelet factor 4 (PF4), FV, fibrinogen-α/γ, prothrombin, protein S, tissue factor pathway inhibitor (TFPI), plasminogen, α−2-macroglobulin, complement C3, C4B, and C9. LDL factions have VWF, platelet glycoprotein-1b-α (GP1bα), PF4, FXIII-α, fibrinogen-α/β/γ, prothrombin, TFPI, plasminogen, complement C3, C4B, and C9. HDL fractions have PF4, FXII, fibrinogen-α/β/γ, prothrombin, antithrombin-III and plasminogen. Lp(a) fractions have VWF, fibrinogen-α/β/γ, FXIII-β, plasminogen, kallikrein, complement C2/C3/C5, C6/C7/C9, and C4B^17–23.

Figure 2. Hemostasis-related proteins identified in human lipoprotein fractions by proteomic studies.

This figure summarizes the findings from several proteomic studies that isolated lipoproteins from human plasma or serum, revealing the presence of various hemostasis-related proteins. Notably, FXIII, prothrombin, fibrinogen, and plasminogen were detected across all lipoprotein fractions, including VLDL, LDL, Lp(a), and HDL. However, it is important to note that the absence of certain proteins in this figure does not necessarily imply a lack of association with lipoproteins due to limited sensitivity by mass spectrometry, particularly for proteins with low plasma concentrations.

The proteins on lipoproteins are not always consistent. Many factors contribute to the variation: 1) the physiological conditions in which the blood samples are collected; 2) how lipoproteins are isolated; and 3) how lipids are removed before analyzing protein content. Due to similar density between Lp(a) and HDL, and similar size between Lp(a) and LDL, without further purification, Lp(a) contamination is in HDL fractions isolated by centrifugation, and in LDL fractions isolated by fast protein liquid chromatography (FPLC)-size exclusion chromatography. In proteomic studies of lipoproteins isolated by size exclusion-FPLC and density gradient ultracentrifugation, protein S, prothrombin, and fibrinogen-γ were found on VLDL and LDL fractions^19,21. In contrast, lipoproteins isolated by density gradient followed by lower-speed centrifugation, protein S, prothrombin, and fibrinogen-γ were found only on VLDL fractions¹⁸. However, most studies did not report the donors’ age, gender, and metabolic information, which contribute to the variation. Further investigations are required to understand how lipoprotein protein contents vary in dyslipidemia in humans.

LDL-apheresis is a procedure to remove circulating apoB-lipoproteins, including VLDL, LDL and Lp(a), from patients with severe hypercholesterolemia that is resistant to lipid-lowering therapies, such as familial hypercholesterolemia (FH). In addition to reducing cholesterol and triglyceride, LDL-apheresis therapy also lowers clotting-related proteins carried on apoB-lipoproteins²⁴. Fibrinogen and TF decreased by 66% and 27%, respectively, in 22 coronary artery disease (CAD) patients undergoing heparin-mediated extracorporeal LDL-precipitation (HELP)-apheresis²⁵. PAI-1 decreased by 72%, 58%, and 30% in three FH patients who received six consecutive LDL-apheresis treatments, respectively²⁶. Another study of nine patients with severe FH, all received LDL-apheresis using either immunoadsorption apheresis (IMA) method (n=5), or dextran-sulfate absorption (DSA) method (n=4). Regardless of the LDL-apheresis method used, all patients had decreased fibrinogen, FV, FVII, FVIII, VWF, FXI, FXII, prekallikrein, antithrombin III, protein C, protein S, α−2-macroglubolin, plasminogen, and PAI-1, with the highest reduction in FVIII by 72% after IMA, and by 98.5% by DSA²⁷. Although many hemostasis-related proteins are present in both apoB-lipoprotein fractions and HDL fractions, the mechanism of how these proteins are associated with lipoproteins and the functional impact of the associate remain unexplored. The different core apolipoproteins and lipids content may impact lipoprotein function and carrying capacity. In the case of HDL, its high radius of curvature enables hydrophobic stretches of plasma proteins to weakly insert²⁸. Therefore, the mechanism contributing to the interaction between the hemostasis proteins and different types and sizes of lipoproteins are likely varied.

3.2. Pro-coagulant roles of phospholipids on lipoproteins

Lipoprotein is the major carrier of circulating neutral phospholipid, such as phosphatidylcholine (PC)²⁹. Coagulation cascade requires an appropriate surface area, such as that provided by phospholipid vesicles from platelets, which reduces the K_m of prothrombinase for prothrombin by 1400-fold^30–33. Lipoproteins do not contain the negatively charged phosphatidylserine (PS), which is essential for coagulation cascade. Normally, PS is located on the inner leaflet of the cell membrane. During platelet activation and cellular apoptosis, PS flips to the outer leaflet of the membrane (inside out). When PS is exposed on the outer membrane of a platelet during blood clotting, the negatively charged phosphatidylserine provides a surface that supports coagulation cascade and promotes clot formation³⁴. Instead, the surface of lipoproteins is made of a monolayer of PC³⁵. Experimental evidence showed VLDL promotes clotting by providing physiological surfaces, abundant in phospholipids, to support extrinsic pathway³⁶. In the presence of prothrombinase complex, VLDL yields thrombin at a rate six-time slower than synthetic phosphatidylcholine/phosphatidylserine (PCPS) vesicles. However, the binding affinity between prothrombin and VLDL is slightly higher than that between prothrombin and PCPS vesicles³⁶.

The oxidation of LDL and phospholipids begins when free radicals, which are highly reactive molecules with unpaired electrons, attack the unsaturated fatty acids present in LDL and phospholipid³⁷. These oxidized LDL (oxLDL) and oxidized PCs are accumulated in atherosclerotic plaques³⁸. Recent studies showed oxidized PCs are also capable of enhancing thrombin generation³⁹. In humans, elevated oxidized PC in apoB-lipoproteins is associated with higher cardiovascular risk⁴⁰. Additionally, oxLDL induce platelet aggregation through multiple mechanism^41–43, which will be discussed in the following sections. However, whether oxidized PCs in apoB-lipoprotein directly interact with coagulation factors is undetermined and requires future investigation.

3.3. Functional impact of lipoproteins and hemostatic system on each other

Although lipoproteins may play a role in hemostasis, the mechanisms by which they serve as hemostatic regulators are poorly understood. Pioneer studies have shed lights on potential function of lipoprotein in association with these clotting-, or clot lysis-related proteins (Figure 3). Hepatocytes is the main source of circulating lipoproteins. Our previous studies found that hepatocytes also synthesize tPA^44,45. Moreover, our recent findings reveal a novel role of intracellular tPA-PAI-1 interaction in determining VLDL assembly and production in hepatocytes⁴⁶. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing VLDL assembly and plasma apoB-lipoprotein cholesterol levels. PAI-1 sequesters tPA away from apoB and increases VLDL assembly. Our study establishes a link between tPA, PAI-1, and plasma levels of atherogenic apoB lipoproteins, beyond the well-known roles of tPA and PAI-1 in the occlusive thrombus that is the ultimate manifestation of atherosclerotic vascular disease⁴⁶. These findings provide a possible mechanistic explanation behind the correlation between high apoB cholesterol and low tPA in ASCVD patients.

Figure 3. Functional impact of lipids and fibrinolysis on each other.

Plasminogen promotes ABCA1-dependent cholesterol efflux to export cholesterol out of macrophages. Both Lp(a) and plasminogen have serine protease domains, while the one in Lp(a) is inactive. Lp(a) also inhibits the conversion of plasminogen to plasmin. Therefore Lp(a) is anti-fibrinolytic. Beyond the well-known roles of tPA in initiating fibrinolysis, and PAI-1 as tPA’s serpin protease inhibitor, we found that tPA interacts with apoB in the haptocytes and blocks apoB lipidation and VLDL production. PAI-1 sequesters tPA away from apoB and increases VLDL assembly.

Plasminogen, found in VLDL, LDL, HDL, and Lp(a) fractions, promotes ABCA1-dependent cholesterol efflux capacity (CEC), transferring cholesterol out of macrophages. Plasminogen-promoted ABCA1-mediated CEC is independent of apoA1, HDL’s core apolipoprotein⁴⁷, but can be inhibited by Lp(a)⁴⁷. Both Lp(a) and plasminogen have serine protease domains, while the one in Lp(a) is inactive, followed by Kringle domain repeats⁴⁸. Lp(a) inhibits the conversion of plasminogen to plasmin, and therefore inhibits fibrinolysis and exacerbates thrombosis^49,50. Moreover, the Kringle domains have abundant lysine-binding sites, allowing Lp(a) to promote coagulation by inhibiting an anticoagulant, TFPI, through blocking its lysine residues in the carboxy-terminus^51,52. LDL also promotes thrombosis by enhancing VWF self-association and subsequent platelet adhesion, leading to increased size and persistence of VWF-platelet thrombi⁵³. In contrast, HDL, or apoA1, prevents VWF self-association and adsorption onto vessel walls, inhibiting platelet activation and adhesion^54,55. HDL also enhance the anticoagulant activity of protein C and protein S⁵⁶. These findings show reciprocal regulation between these two systems (Table 1).

Table 1.

Effect of lipids or lipoproteins on hemostasis-related proteins

	Clinical observations	Mechanistic studies
VWF	Obesity and dyslipidemia: associates with higher VWF83,91,153	LDL: promotes VWF self-association154 HDL: inhibits VWF self-association54
Platelet	LDL cholesterol: associateswith higher platelet count70,71 HDL cholesterol: associates with lower platelet count70,76 Total cholesterol/triglycerides: associates with higher platelet count70,71, shorter platelet life span74,75 and higher platelet activation77–79	oxLDL: induces platelet activation through CD3642 HDL/apoA1: supresses platelet production through increasse cholesterol efflux in megakaryocytes progenitor cells68,69; reduces platelet activation and aggregation55,68,82
Coagulation	Obesity: associates with higher monocyte TF activity86, higher FVII84,153, higher thrombin85, and denser clots87,89. High triglyceride: associates with higher activation of platelets92, higher FVII and FX93	oxLDL: increases TF expression in monocytes/macrophages106,107 and in vascular smooth muscle cells110 Lp(a): inhibits TFPI through blocking its lysine residues in the carboxy-terminus51,52 HDL: enhances the anticoagulant activities of protein C and protein S56 Obesity: higher TF in obese mice105 Cholesterol: increases TF expression in monocytes/macrophages106–109; decreases TF activity by depletion of cholesterol in human fibroblast111
Fibrinolysis	Obesity: associates with higher PAI-1129,130, higher tPA135 High triglyceride: higher PAI-1130,131	VLDL: stimulates PAI-1 secretion in humann umbilical vein endothelial cells or HepG2 cells133,134 Lp(a): inhibits conversion of plasminogen to plasmin by tPA147,149 Obesity: higher PAI-1 and tPA44, lower tPA activity44 in obese mice Fatty acids: increases hepatocyte PAI-144 and tPA44, decreases hepatocyte PAI-1 free tPA44

4. Thrombotic tendency in obesity and dyslipidemia

Obesity has become a pandemic, increasing risks for arterial, venous, and microvascular thrombosis, including MI, IS, venous thromboembolism (VTE) and thrombotic thrombocytopenic purpura⁵⁷. 70% of obese people have dyslipidemia⁵⁸, which is independently associated with thrombosis^59–61, suggesting dyslipidemia could be one of the causal mechanisms driving thrombosis in obesity. Obesity-associated dyslipidemia is characterized by higher plasma apoB-lipoproteins cholesterol and triglyceride, due to increased hepatic and intestinal apoB-lipoprotein production^62,63, decreased apoB-lipoprotein clearance, and reduced HDL cholesterol⁶⁴. The cholesteryl ester transfer protein (CETP) facilitates transfer of cholesteryl esters from HDL to apoB-lipoproteins. Increased CETP activities and protein mass have been observed in obese subjects, contributing to a decrease in HDL cholesterol levels⁶⁵.

Chylomicron and VLDL contain 90% and 60% triglycerides, respectively¹⁶. These lipoproteins are assembled in intestinal epithelial cells and liver hepatocytes to transport triglyceride to other organs and tissues. Dietary lipid-overloading to the intestinal epithelial cells and hepatocytes promotes the assembly and secretion of chylomicrons and VLDL to circulation, leading to higher circulating apoB-lipoproteins, triglyceride, and cholesterol^66,67.

4.1. Increased platelet count, activation and aggregation in dyslipidemia

Cholesterol homeostasis has impacts on hematopoiesis, platelet biogenesis and platelet activation. Megakaryocyte progenitors give rise to megakaryocytes during hematopoiesis (megakaryopoiesis) in the bone marrow. ATP-Binding Cassette Subfamily G 4 (ABCG4) is a transporter which facilitates cholesterol efflux to HDL and is expressed in megakaryocytes progenitor cells^68,69. ABCG4 deficiency led to defective cholesterol efflux to HDL and increased free cholesterol accumulation in megakaryocytes progenitor cells in association with increased megakaryopoiesis and higher platelet counts⁶⁸. Moreover, infusion of rHDL reduced megakaryocytes progenitor cells proliferation and platelet counts in wild-type mice, but not in Abcg4^−/− mice⁶⁸. Taken together, those findings indicate HDL/ABCG4-mediated cholesterol efflux in megakaryocytes progenitor cells suppresses megakaryocytes and platelet production⁶⁸. However, whether apoB lipoproteins have an impact on megakaryopoiesis is undetermined and is worthy of investigating in future studies.

Platelets are budding off from megakaryocytes through a process called platelet biogenesis. Specifically, megakaryocytes extend proplatelet pseudopodia into blood vessels, where shear forces break off small fragments that become platelets. Plasma LDL cholesterol is positively correlated with platelet counts in humans^70,71. The findings in humans have been recapitulated in animal studies which show diet-induced hypercholesterolemia led to thrombocytosis in mice⁷². However, hypercholesterolemia is also associated with shortened platelet lifespan and increased turnover rate^73–75. Therefore, these findings suggest the net increased platelet numbers in hypercholesterolemia is due to enhanced platelet biogenesis, which exceeds the shorter platelet lifespan and clearance rate. In contrast, HDL cholesterol levels are negatively associated with platelet count in humans⁷⁶, and infusion of rHDL reduced platelet counts in mice⁶⁸.

FH, a genetic disorder characterized by high levels of LDL cholesterol, is associated with an abnormal platelet function, including higher platelet activation, platelet-releasing 12-hydroxyeicosatetraenoic acid and thromboxane B2, cell-free nucleotide, impaired vasodilator responses and enhanced platelet aggregation^77–80. Hypercholesterolemia increases platelet activation through multiple mechanisms. Increased oxLDL and oxidized-phosphatidylserine (oxPS) in hypercholesterolemia induce platelet activation through the engagement of their receptor CD36⁴². This process leads to the inhibition of NO synthase⁴³, activation of phospholipase A2⁴¹ and surface expression of activated GPIIb/IIIa and P-selectin⁸¹. Contrary to the role of LDL cholesterol, which increase platelet activity, the infusion of reconstitute-HDL (rHDL) in individuals with type 2 diabetes mellitus is associated with > 50% reduction in platelet aggregation⁸². The depletion of cholesterol in platelet membranes via HDL-dependent cholesterol efflux mechanism may contribute to the reduced platelet aggregation by rHDL.

4.2. Hypercoagulability in obesity and dyslipidemia

The connection between thrombosis and obesity, or dyslipidemia, also involves elevated levels of the prothrombotic molecules. Obese or dyslipidemia patients have higher plasma concentrations of VWF⁸³, FVII⁸⁴, thrombin⁸⁵, circulating monocyte TF procoagulant activity⁸⁶, and also have denser clots, packed with thin fibers that are lysed slower than the light and loosely packed fibrin clots in non-obese patients^87–90. Higher serum triglyceride levels also associate with a higher risk of thrombosis, higher VWF levels⁹¹, and higher activation of platelets, FVII and FX^92,93.

TF is ubiquitously expressed across many organs and various cell types, including smooth muscle cells in blood vessels, astrocytes in the brain, epithelial cells encompassing organs, cardiomyocytes in the heart, and circulating monocytes⁹⁴. High levels of TF have been detected in brain, lung, and placenta; moderate levels in heart, kidney, intestine, testes and uterus; and lower levels in the spleen, thymus and liver⁹⁴. TF is known to play a critical role as the primary cellular initiator of coagulation cascade. Deficiencies in TF is lethal in mice, and there is no reported instances of human TF deficiency⁹⁵.

High TF level is associated with intravascular thrombosis in various diseases, such as sepsis, cancer, and atherosclerosis^96–98. Several studies reported high levels of TF antigen in human atherosclerotic plaque^99–102. TF is higher in lesions from patients with unstable coronary syndrome compared to patients with stable coronary syndrome¹⁰¹. These findings suggest TF contributes to the atherothrombosis events. Interestingly, cholesterol also induces TF expression. Patients with hypercholesterolemia showed higher TF levels in circulating monocytes compared to the control subjects^103,104. Similarly, in obese mice, adipose TF mRNA is upregulated and associates with the increased circulating TF¹⁰⁵. Experimental evidence demonstrates both cholesterol loading and incubating ox-LDL with monocytes or macrophages result in increased TF expression^106–109. Lysophosphatidic acid, which is a component of oxidized lipoprotein, increased TF expression and activity in vascular smooth muscle cells prepared from explants of excised aortas of rats¹¹⁰. In contrast, depletion of membrane cholesterol of human fibroblasts with methyl-β-cyclodextrin reduced TF activity, while treating the cholesterol-depleted cells with cholesterol restoring TF expression and activity¹¹¹. However, the underlying mechanism remains uncertain and requires further investigation.

Coagulopathies are frequently seen in severe SARS-CoV-2 virus infections^112–114 with both arterial and venous thrombosis outcomes, including MI, pulmonary embolism, and deep vein thrombosis^115,116. The incidence of thrombotic and thromboembolic complications in patients with moderate and severe COVID-19 ranges from 21% to 49%, while even higher in non-surviving patients^{112,117–119}. Development of hypertriglyceridemia is independently correlated with thrombosis incidence and inpatient mortality in those with severe COVID-19 infection, after adjusting for age, gender, and obesity^120,121. Lp(a) is a risk factor for thrombotic cardiovascular disease, including myocardial infarction and ischemic stroke^122–125. In patients with COVID-19, higher Lp(a) was reported to be positively associated with ischemic heart disease and VTE^126,127. Our recent study also showed higher Lp(a) in hospitalized COVID-19 patients with high tPA antigen level and low tPA enzymatic activity level¹²⁸. Further research regarding the mechanism behind severe COVID-19 with dyslipidemia and thrombotic outcomes is needed.

4.3. Impaired fibrinolysis in obesity and dyslipidemia

Among the myriad metabolic abnormalities related to obesity, impaired fibrinolysis has been recognized as a mechanistic pathway for obesity-associated thrombosis. tPA initiates fibrinolysis by catalyzing the conversion of the inactive zymogen plasminogen into plasmin, which is a serine protease responsible for breaking down fibrin clots¹⁴. PAI-1, a fast-acting suicidal serpin (serine protease inhibitor), inactivates tPA by forming a complex with it, leading to the inhibition of the fibrinolysis¹⁵. Total PAI-1 antigen is higher in obese patients^129,130, or those with high triglyceride levels^130–132. Incubating human umbilical vein endothelial cells or HepG2 hepatoma cells with purified human VLDL stimulates PAI-1 secretion^133,134. Although tPA also increased in obesity¹³⁵, the increase of PAI-1 is higher than tPA in obesity, leading to a net reduction in tPA activity and fibrinolytic potential, delaying fibrin clot removal. Therefore, it is the PAI-1-free tPA levels that reflect fibrinolytic potential¹³⁶. In dyslipidemia, tPA antigen is positively correlated with PAI-1¹³⁷, suggesting compensatory feedback when there is insufficient active tPA. Furthermore, plasma tPA antigen level is negatively associated with tPA activity shown by several independent studies^{128,138–140}. These observations suggest that impaired fibrinolysis could be a link between dyslipidemia and an increased risk of thrombosis.

Our previous studies revealed that hepatocytes are an unappreciated source of tPA that contribute ~40–50% plasma basal tPA concentration, which is important for fibrinolysis when a vessel injury occurs^44,45. We further investigated how hepatocytes sense metabolic stresses and imbalance of production of tPA and its serpin inhibitor PAI-1⁴⁴. Lipid-overloaded hepatocytes have reduced transcription co-repressor Rev-Erbα, leading to increased PAI-1, which then stimulates tPA synthesis via a PAI-1-LRP1-PKA-p-CREB1 pathway⁴⁴. This PAI-1-stimulating tPA production mechanism functions as a compensatory pathway to compete with the large increase of PAI-1. However, the small induction of tPA synthesis is limited by its transcription repressor DACH1, which is induced in obese livers. In hepatocytes, where lipids are loaded and incorporated into lipoproteins, this PAI-1-tPA regulatory network influences the degree of impaired fibrinolysis in obesity.

Another potential link between impaired fibrinolysis and dyslipidemia is Lp(a)¹⁴¹. Lp(a) contains one LDL-like particle and one apolipoprotein(a), (apo(a)). Apo(a) is encoded by LPA gene, which is only expressed in primates¹⁴². This gene is evolved from the plasminogen gene through duplication and remodeling over millennia, leading to the structural similarity between Lp(a) and plasminogen¹⁴². Like other apoB-lipoproteins, Lp (a) also carries cholesterol^141,143. Clinical studies demonstrate that Lp(a) levels are associated with ASCVD risk quantitatively¹⁴⁴. Plasma Lp(a) level is genetically determined, with approximately 25% of population having high Lp(a) greater than 30 mg/dL¹⁴⁴. People with Lp(a) higher than 30 mg/dL have two times higher risk in CADs¹⁴⁵. Lp(a) becomes a significant contributor to the plasma total-cholesterol and the risk of ASCVD when its levels rise above 30 mg/dL¹⁴⁶. Unlike plasminogen, apo(a) cannot be converted to an active protease because a serine substitution to arginine at the position that can be cleaved by tPA^147,148. Therefore, Lp(a) competes with plasminogen and inhibits fibrinolysis by binding to lysine binding sites within Kringle IV domains of Lp(a), thereby increasing thrombosis risk^147,149. In addition to inhibit fibrinolysis, the Women’s Healthy study with 25,131 Caucasian women participants, showed that individuals with high Lp(a) levels have greater benefits in lowering atherothrombotic events by low dose aspirin compared to those with low Lp(a) levels (median Lp(a), 153.9 versus 10.0 mg/dL)¹⁵⁰. Over the 9.9 years of follow-up, the use of aspirin reduced cardiovascular risk in individuals with high Lp(a) levels (age-adjusted hazard ratio =0.44, 95% CI: 0.20–0.94), but not in those with low Lp(a) (HR=0.91, 95% CI: 0.77–1.08). The potential mechanisms underlying this effect are not yet clear and require validation.

5. Conclusion

As summarized in this review, lipoproteins are associated with proteins related to blood clotting by proteomics. Obesity or dyslipidemia is associated with impaired fibrinolysis and hypercoagulable tendencies and increased risk of ischemic stroke, deep vein thrombosis, and pulmonary embolism in men and women across all ethnic groups^151,152. However, the distribution of specific risk factors mentioned above is poorly studied in all ethnicity groups and requires further investigation. More studies are required to understand the link between lipoproteins and the hemostatic system regarding the functional impact of lipoprotein-associated clotting factors on hemostasis and thrombosis, and strategies to improve clinical care to balance the risk of thrombosis and bleeding.

Highlights.

• Clotting-related proteins are associated with lipoproteins by proteomics.

• LDL-apheresis reduces clotting-related protein levels in plasma.

• Dyslipidemia is associated with increased platelet count, platelet activity, hypercoagulability, and impaired fibrinolysis.

Nonstandard abbreviations and acronyms

AF

Atrial fibrillation

ASCVDs

Atherosclerotic cardiovascular diseases

ABCG4

ATP-Binding Cassette Subfamily G4

Apo(a)

Apolipoprotein(a)

aPTT

Activated partial thromboplastin time

CAD

Coronary artery disease

CEC

Cholesterol efflux capacity

CETP

Cholesteryl ester transfer protein

DSA

Dextran-sulfate absorption

FI

Factor I

FII

Factor II

FIIa

Activated factor II

FV

Factor V

FVa

Activated factor V

FVII

Factor VII

FVIIa

Activated factor VII

FVIII

Factor VIII

FVIIIa

Activated factor VIII

FIX

Factor IX

FIXa

Activated factor IX

FX

Factor X

FXa

Activated factor X

FXI

Factor XI

FXIa

Activated factor XI

FXII

Factor XII

FXIIa

Activated factor XII

FXIII

Factor XIII

FXIIIa

Activated factor XIII

PF4

Platelet factor 4

FPLC

Fast protein liquid chromatography

FH

Familial hypercholesterolemia

GP1bα

Glycoprotein-1b-α

HDL

High-density lipoprotein

HELP

Heparin-mediated extracorporeal LDL-precipitation

IDL

Intermediate-density lipoprotein

IMA

Immunoadsorption apheresis

LDL

Low-density lipoprotein

Lp(a)

Lipoprotein(a)

MI

Myocardial infarction

MTP

Microsomal triglyceride transfer protein

NHANES

National Health and Nutrition Examination Survey

oxLDL

Oxidized LDL

oxPS

Oxidized phosphatidylserine

PAI-1

Plasminogen activator inhibitor 1

PC

Phosphatidylcholine

PS

Phosphatidylserine

PCPS

Phosphatidylcholine/phosphatidylserine

PT

Prothrombin time

rHDL

Reconstitute-HDL

RCTs

Randomized controlled trials

TF

Tissue factor

TFPI

Tissue factor pathway inhibitor

tPA

Tissue-type plasminogen activator

uPA

Urokinase plasminogen activator

VWF

Von Willebrand factor

VLDL

Very-low-density lipoprotein

VTE

Venous thromboembolism