Protease-Activated Receptor 1 Contributes to Angiotensin II-Induced Cardiovascular Remodeling and Inflammation.

BACKGROUND: Angiotensin II (Ang II) plays an important role in cardiovascular disease. It also leads to the activation of coagulation. The coagulation protease thrombin induces cellular responses by activating protease-activated receptor 1 (PAR-1). We investigated whether PAR-1 contributes to Ang II-induced cardiovascular remodeling and inflammation. METHODS AND RESULTS: PAR-1+/+ (wild-type; WT) and PAR-1-/- mice were infused with Ang II (600 ng/kg/min) for up to 4 weeks. In WT mice, this dose of Ang II did not cause a significant increase in blood pressure but it did cause pathological changes in both the aorta and the heart. Ang II infusion resulted in vascular remodeling of the aorta, demonstrated by a significant increase in medial wall thickening and perivascular fibrosis. Importantly, both parameters were significantly attenuated by PAR-1 deficiency. Furthermore, perivascular fibrosis around coronary vessels was reduced in Ang II-treated PAR-1-/- mice compared to WT mice. In addition, PAR-1 deficiency significantly attenuated Ang II induction of inflammatory cytokines and profibrotic genes in the aortas compared to WT mice. Finally, PAR-1 deficiency had no effect on Ang II-induced heart hypertrophy. However, the heart function measured by fractional shortening was less impaired in PAR-1-/- mice than in WT mice. CONCLUSION: Our data indicate that PAR-1 plays a significant role in cardiovascular remodeling mediated by a blood pressure-independent action of Ang II.

Emerging pathophysiological roles for fibrinolysis.

PURPOSE OF REVIEW: The fibrinolytic system plays a key role in the regulation of hemostasis and thrombosis; however, it also has multiple pleiotropic effects. In this review, we examine the studies that investigated the role of the plasminogen activator system and its inhibitors outside the context of clot lysis. RECENT FINDINGS: Activators of plasminogen, plasminogen receptors, and inhibitors of plasminogen activation all play a role in the proliferation, migration, and metastasis of tumor cells in many cancer types and may serve as prognostic and diagnostic markers. The plasminogen activator system is also involved in the pathogenesis and severity of several inflammatory diseases, including sepsis, metabolic disease, arthritis, and airway disease. A study on the use of tissue plasminogen activator (tPA) following cerebrovascular events demonstrates that tPA also plays important roles in the pathogenesis of stroke and affects the long-term outcomes. SUMMARY: Current evidence suggests an association between the plasminogen activator system and its inhibitors in a variety of malignant and inflammatory states. Newly discovered roles for plasminogen activators and plasminogen activator inhibitors in these diseases provide novel targets for future therapeutic development. Additionally, the newly characterized regulation of the plasminogen activator system by endogenous microRNAs provides new insight into the physiological role of this system and its role in disease.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer.

Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Causal relationship between hyperfibrinogenemia, thrombosis, and resistance to thrombolysis in mice.

Epidemiologic studies have correlated elevated plasma fibrinogen (hyperfibrinogenemia) with risk of cardiovascular disease and arterial and venous thrombosis. However, it is unknown whether hyperfibrinogenemia is merely a biomarker of the proinflammatory disease state or is a causative mechanism in the etiology. We raised plasma fibrinogen levels in mice via intravenous infusion and induced thrombosis by ferric chloride application to the carotid artery (high shear) or saphenous vein (lower shear); hyperfibrinogenemia significantly shortened the time to occlusion in both models. Using immunohistochemistry, turbidity, confocal microscopy, and elastometry of clots produced in cell and tissue factor-initiated models of thrombosis, we show that hyperfibrinogenemia increased thrombus fibrin content, promoted faster fibrin formation, and increased fibrin network density, strength, and stability. Hyperfibrinogenemia also increased thrombus resistance to tenecteplase-induced thrombolysis in vivo. These data indicate that hyperfibrinogenemia directly promotes thrombosis and thrombolysis resistance and does so via enhanced fibrin formation and stability. These findings strongly suggest a causative role for hyperfibrinogenemia in acute thrombosis and have significant implications for thrombolytic therapy. Plasma fibrinogen levels may be used to identify patients at risk for thrombosis and inform thrombolytic administration for treating acute thrombosis/thromboembolism.

Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis.

The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis.

Overexpression of the cell cycle inhibitor p16INK4a promotes a prothrombotic phenotype following vascular injury in mice.

OBJECTIVE: Age-associated cellular senescence is thought to promote vascular dysfunction. p16(INK4a) is a cell cycle inhibitor that promotes senescence and is upregulated during normal aging. In this study, we examine the contribution of p16(INK4a) overexpression to venous thrombosis. METHODS AND RESULTS: Mice overexpressing p16(INK4a) were studied with 4 different vascular injury models: (1) ferric chloride (FeCl(3)) and (2) Rose Bengal to induce saphenous vein thrombus formation; (3) FeCl(3) and vascular ligation to examine thrombus resolution; and (4) lipopolysaccharide administration to initiate inflammation-induced vascular dysfunction. p16(INK4a) transgenic mice had accelerated occlusion times (13.1 ± 0.4 minutes) compared with normal controls (19.7 ± 1.1 minutes) in the FeCl(3) model and 12.7 ± 2.0 and 18.6 ± 1.9 minutes, respectively in the Rose Bengal model. Moreover, overexpression of p16(INK4a) delayed thrombus resolution compared with normal controls. In response to lipopolysaccharide treatment, the p16(INK4a) transgenic mice showed enhanced thrombin generation in plasma-based calibrated automated thrombography assays. Finally, bone marrow transplantation studies suggested increased p16(INK4a) expression in hematopoietic cells contributes to thrombosis, demonstrating a role for p16(INK4a) expression in venous thrombosis. CONCLUSIONS: Venous thrombosis is augmented by overexpression of the cellular senescence protein p16(INK4a).

Mature breast adipocytes promote breast cancer cell motility.

Adipocytes express substances involved in both normal physiology and pathological processes. One such adipocyte protein is the Serpin (serine protease inhibitor) plasminogen activator inhibitor-1 (PAI-1). PAI-1 functions to inhibit urokinase type plasminogen activator (uPA) though PAI-1 itself is also implicated in breast cancer progression. While the role of adipocytes in breast cancer development is not fully understood, obesity is a known risk factor associated with breast cancer. Thus, we characterized adipocytes from breast and omental tissues for PAI-1 and uPA, and the influence of adipocytes on breast cancer cell motility. Using preadipocyte cells from breast and omental adipose tissue, we differentiated each site into mature adipocytes. PAI-1 protein was found in breast adipocytes>omental preadipocytes>omental adipocytes>breast preadipocytes. Interestingly, uPA protein was not detected in any of these cell types. We then incubated breast adipocyte conditioned media (Adip-CM) and preadipocyte conditioned media (PreAdip-CM) on both normal (MCF-10A) and malignant (MCF-10CA1) breast epithelial cell lines. Adip-CM, but not PreAdip-CM, (a) increased cell motility in both MCF-10A and MCF-10CA1 cells; (b) increased cell-associated uPA activity in both cell lines; (c) increased phosphorylated-Akt levels in MCF-10CA1 cells; and (d) gene array profiles show altered expression of several genes associated with cancer adhesion, metastasis and signaling. Our results suggest that mature breast adipocytes are capable of altering the epithelial cell phenotype, producing a more motile cell type and further provide a potential link between obesity and risk of breast cancer.

Suggestions on leading an academic research laboratory group.

This commentary is about running an academic research laboratory group, including some reflections, memories, and tips on effectively managing such a group of scientists focused on one's research. The author's academic career has spanned from 1982 to 2022, including postdoctoral research associate through the rank of professor with tenure. Currently, the author is in the final year of 3 years of phased retirement. One must be willing to work hard at running a research laboratory. Also, stay focused on funding the laboratory tasks and publishing one's work. Recruit the best people possible with advice from the collective laboratory group. Laboratory group members felt more like they were a part of a collective family than simply employees; however, what works best for the researcher is what matters. Several other points to discuss will include managing university roles, recruiting laboratory personnel, getting recognition, dealing with intellectual property rights, and publishing work. In closing, there are many more positives than negatives to leading a research laboratory group. Finally, one cannot replace the unforgettable memories and the legacy of a research laboratory group.

Role of SARS-CoV-2 in Modifying Neurodegenerative Processes in Parkinson's Disease: A Narrative Review.

The COVID-19 pandemic, caused by SARS-CoV-2, continues to impact global health regarding both morbidity and mortality. Although SARS-CoV-2 primarily causes acute respiratory distress syndrome (ARDS), the virus interacts with and influences other organs and tissues, including blood vessel endothelium, heart, gastrointestinal tract, and brain. We are learning much about the pathophysiology of SARS-CoV-2 infection; however, we are just beginning to study and understand the long-term and chronic health consequences. Since the pandemic's beginning in late 2019, older adults, those with pre-existing illnesses, or both, have an increased risk of contracting COVID-19 and developing severe COVID-19. Furthermore, older adults are also more likely to develop the neurodegenerative disorder Parkinson's disease (PD), with advanced age as the most significant risk factor. Thus, does SARS-CoV-2 potentially influence, promote, or accelerate the development of PD in older adults? Our initial focus was aimed at understanding SARS-CoV-2 pathophysiology and the connection to neurodegenerative disorders. We then completed a literature review to assess the relationship between PD and COVID-19. We described potential molecular and cellular pathways that indicate dopaminergic neurons are susceptible, both directly and indirectly, to SARS-CoV-2 infection. We concluded that under certain pathological circumstances, in vulnerable persons-with-Parkinson's disease (PwP), SARS-CoV-2 acts as a neurodegenerative enhancer to potentially support the development or progression of PD and its related motor and non-motor symptoms.

Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration.

BACKGROUND: Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer. METHODS AND RESULTS: We found that PCI inhibits cathepsin L with an inhibition rate (k(2)) of 3.0x10(5)M(-)(1)s(-)(1). Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells. CONCLUSIONS: Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L. GENERAL SIGNIFICANCE: Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.

Heparin cofactor II: discovery, properties, and role in controlling vascular homeostasis.

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) found in high concentrations in human plasma. Despite its discovery >30 years ago, its physiological function is still poorly understood. It is known to inhibit thrombin, the predominant coagulation protease, and HCII-thrombin complexes have been found in plasma, yet it is thought to contribute little to normal hemostasis. However, thrombin has several other physiological functions, and therefore many biological roles for HCII need consideration. The unique structure and mechanism of action of HCII have helped guide our understanding of HCII. In particular, HCII binds many glycosaminoglycans (GAGs) such as heparin and heparin sulfate as well as several different polyanions to enhance its inhibition of thrombin. Distinctly, HCII is able to use the GAG dermatan sulfate for accelerated thrombin inhibition. Dermatan sulfate is found in high concentrations in the walls of blood vessels as well as in placental tissue. This knowledge has led to research indicating that HCII may play a protective role in atherosclerosis and placental thrombosis. Additionally, pharmaceuticals are being developed that use the dermatan sulfate activation of HCII for anticoagulation. Although much research is still needed to fully understand HCII, this humble protein may have significant impact in our medical future. This article reviews the laboratory history, protein characteristics, structure-activity relationships, protease inhibition, physiological function, and medical relevance of HCII in hopes of regenerating interest in this sometimes forgotten serpin.

Activated protein C enhances cell motility of endothelial cells and MDA-MB-231 breast cancer cells by intracellular signal transduction.

Activated protein C (APC), an anticoagulant serine protease, has been shown to have non-hemostatic functions related to inflammation, cell survival, and cell migration. In this study we investigate the mechanism by which APC promotes angiogenesis and breast cancer invasion using ex vivo and in vitro methods. When proteolytically active, APC promotes cell motility/invasion and tube formation of endothelial cells. Ex vivo aortic ring assays verify the role of APC in promoting angiogenesis, which was determined to be dependent on EGFR and MMP activation. Given the capacity of APC to promote angiogenesis and the importance of this process in cancer pathology, we investigated whether the mechanisms by which APC promotes angiogenesis can also promote motility and invasion in the MDA-MB-231 breast cancer cell line. Our results indicate that, extracellularly, APC engages EPCR, PAR-1, and EGFR in order to increase the invasiveness of MDA-MB-231 cells. APC activation of matrix metalloprotease (MMP) -2 and/or -9 is necessary but not sufficient to increase invasion, and APC does not utilize the endogenous plasminogen activation system to increase invasion. Intracellularly, APC activates ERK, Akt, and NFkappaB, but not the JNK pathway to promote MDA-MB-231 cell motility. Similar to the hemostatic protease thrombin, APC has the ability to enhance both endothelial cell motility/angiogenesis and breast cancer cell migration.

Treatment Options for Motor and Non-Motor Symptoms of Parkinson's Disease.

Parkinson's disease (PD) usually presents in older adults and typically has both motor and non-motor dysfunctions. PD is a progressive neurodegenerative disorder resulting from dopaminergic neuronal cell loss in the mid-brain substantia nigra pars compacta region. Outlined here is an integrative medicine and health strategy that highlights five treatment options for people with Parkinson's (PwP): rehabilitate, therapy, restorative, maintenance, and surgery. Rehabilitating begins following the diagnosis and throughout any additional treatment processes, especially vis-à-vis consulting with physical, occupational, and/or speech pathology therapist(s). Therapy uses daily administration of either the dopamine precursor levodopa (with carbidopa) or a dopamine agonist, compounds that preserve residual dopamine, and other specific motor/non-motor-related compounds. Restorative uses strenuous aerobic exercise programs that can be neuroprotective. Maintenance uses complementary and alternative medicine substances that potentially support and protect the brain microenvironment. Finally, surgery, including deep brain stimulation, is pursued when PwP fail to respond positively to other treatment options. There is currently no cure for PD. In conclusion, the best strategy for treating PD is to hope to slow disorder progression and strive to achieve stability with neuroprotection. The ultimate goal of any management program is to improve the quality-of-life for a person with Parkinson's disease.

PRIME Immunology: Self-directed Introduction to Medical School Immunology.

INTRODUCTION: Medical students find immunology difficult to understand and relate to clinically and are often frustrated by the amount of detailed material. We created PRIME Immunology: Preview or Review of Important Material for Everyone: (i) video modules, (ii) Instagram site, and (iii) vocabulary files called Immunology Language. METHODS: The self-paced modules introduced key topics in immunology for students to complete prior to their instructional block. RESULTS AND CONCLUSIONS: Use of PRIME Immunology during a 3-year period suggested that providing students with an overview of key topics before the start of their course may (i) reduce student angst about immunology and (ii) improve retention of immunology.

Golf as a Physical Activity to Potentially Reduce the Risk of Falls in Older Adults with Parkinson's Disease.

Advanced age is associated with an increased risk for falls in aging adults. Older adults are also more likely to be diagnosed with Parkinson's disease (PD), with advanced age as the most significant risk factor. PD is a neurodegenerative disorder with four Cardinal motor symptoms: rigidity, bradykinesia, postural instability, and tremor. Thus, people (person)-with-Parkinson's disease (PwP) have an even greater risk of falling than non-disorder age-matched peers. Exercise is an activity requiring physical effort, typically carried out to sustain or improve overall health and fitness, and it lowers the risk of falls in the general population. The sport of golf provides a low-impact all-around workout promoting a range of motion, activation of muscles in the upper and lower body, flexibility, and balance. Swinging a golf club offers a unique combination of high amplitude axial rotation, strengthening postural musculature, coordination, and stabilization, demonstrating the potential to impact PD symptoms positively. Golf may be a novel exercise treatment regimen for PD to use in conjunction with traditional medical therapy. We completed a literature review to determine the relationship between the game of golf, PD, and the risk of falls. We concluded that regularly playing golf can lower the risk for falls in community ambulating older adults with PD and demonstrates the potential to improve quality of life for PwP.

Exercise for Older Adults Improves the Quality of Life in Parkinson's Disease and Potentially Enhances the Immune Response to COVID-19.

Parkinson's disease (PD) is a progressive neurodegenerative disorder brought about due to dopaminergic neuronal cell loss in the midbrain substantia nigra pars compacta region. PD presents most commonly in older adults and is a disorder of both motor and nonmotor dysfunction. The novel SARS-CoV-2 virus is responsible for the recent COVID-19 pandemic, and older individuals, those with preexisting medical conditions, or both have an increased risk of developing COVID-19 with more severe outcomes. People-with-Parkinson's (PwP) of advanced age can have both immune and autonomic nervous problems that potentially lead to pre-existing pulmonary dysfunction and higher infection risk, increasing the probability of contracting COVID-19. A lifestyle change involving moderate-intensity exercise has the potential to protect against SARS-CoV-2 through strengthening the immune system. In addition to a potential protective measure against SARS-CoV-2, exercise has been shown to improve quality-of-life (QoL) in PD patients. Recent studies provide evidence of exercise as both neuroprotective and neuroplastic. This article is a literature review investigating the role exercise plays in modifying the immune system, improving health outcomes in PwP, and potentially acting as a protective measure against SARS-Cov-2 infection. We conclude that exercise, when correctly performed, improves QoL and outcomes in PwP, and that the enhanced immune response from moderate-intensity exercise could potentially offer additional protection against COVID-19.

Synergy between plasminogen activator inhibitor-1, α-synuclein, and neuroinflammation in Parkinson's disease.

Parkinson's disease (PD) is a progressive degenerative nervous system disorder and is the second most common neurodegenerative disorder in the elderly population. The disease originates from the loss of dopamine-producing neurons in the substantia nigra in the brain, resulting in unregulated activity of the basal ganglia. Αlpha-synuclein (α-syn) is a protein found to aggregate in the substantia nigra region of patients with PD, forming Lewy Body inclusions; its aggregation may contribute to neuronal cell death in PD. This work hypothesizes about the synergistic relationship between α-syn aggregation and neuroinflammation to up-regulate expression of the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1). The protease, plasmin, has been shown to cleave extracellular α-syn (including its monomeric, oligomeric, and fibrillary forms), resulting in less aggregation and Lewy Body formation. The zymogen plasminogen is converted to its active serine protease form, plasmin, either by tissue plasminogen activator (tPA) or by urokinase plasminogen activator (uPA) bound to urokinase receptor (uPAR). Both tPA and uPA/uPAR are inhibited by PAI-1. Thus, when PAI-1 levels increase, less plasmin is generated, which would lead to reduced proteolysis of α-syn. Expression of PAI-1 is increased both in inflammatory environments and in the presence of extracellular α-syn aggregates. This scenario suggests a pathological amplification loop: increased extracellular α-syn aggregation activates an inflammatory response from microglia and astrocytes, increasing PAI-1 levels, and decreasing the generation of plasmin. With reduced plasmin, less α-syn can be cleaved, and aggregation continues, sustaining the pathological process. Understanding this putative pathogenic loop could provide insight into the means by which neurodegeneration progresses in PD, and it may offer possible novel therapeutic strategies.

Potential Role of Vitamin D in the Elderly to Resist COVID-19 and to Slow Progression of Parkinson's Disease.

While we are still learning more about COVID-19, caused by the novel SARS-CoV-2 virus, finding alternative and already available methods to reduce the risk and severity of the disease is paramount. One such option is vitamin D, in the form of vitamin D3 (cholecalciferol) supplementation, due to its potential antiviral properties. It has become apparent that older individuals have a greater risk of developing severe COVID-19, and compared to younger adults, the elderly have lower levels of vitamin D due to a variety of biological and behavioral factors. Older adults are also more likely to be diagnosed with Parkinson's disease (PD), with advanced age being the single greatest risk factor. In addition to its immune-system-modulating effects, it has been suggested that vitamin D supplementation plays a role in slowing PD progression and improving PD-related quality of life. We completed a review of the literature to determine the relationship between vitamin D, PD, and COVID-19. We concluded that the daily supplementation of 2000-5000 IU/day of vitamin D3 in older adults with PD has the potential to slow the progression of PD while also potentially offering additional protection against COVID-19.

Heparin cofactor II in atherosclerotic lesions from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study.

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) that has been shown to be a predictor of decreased atherosclerosis in the elderly and protective against atherosclerosis in mice. HCII inhibits thrombin in vitro and HCII-thrombin complexes have been detected in human plasma. Moreover, the mechanism of protection against atherosclerosis in mice was determined to be the inhibition of thrombin. Despite this evidence, the presence of HCII in human atherosclerotic tissue has not been reported. In this study, using samples of coronary arteries obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, we explore the local relationship between HCII and (pro)thrombin in atherosclerosis. We found that HCII and (pro)thrombin are co-localized in the lipid-rich necrotic core of atheromas. A significant positive correlation between each protein and the severity of the atherosclerotic lesion was present. These results suggest that HCII is in a position to inhibit thrombin in atherosclerotic lesions where thrombin can exert a proatherogenic inflammatory response. However, these results should be tempered by the additional findings from this, and other studies, that indicate the presence of other plasma proteins (antithrombin, albumin, and alpha(1)-protease inhibitor) in the same localized region of the atheroma.

Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition.

Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.