A Representative Clinical Course of Progression, with Molecular Insights, of Hormone Receptor-Positive, HER2-Negative Bone Metastatic Breast Cancer.

Despite treatment advances, breast cancer remains a leading cause of death of women in the United States, mostly due to metastatic disease. Bone is a preferential site for breast cancer metastasis, and most metastatic breast cancer patients experience bone involvement at the time of death. The majority of patients with bone metastatic breast cancer are first diagnosed with and treated for early-stage disease, and from development of early-stage breast cancer to the recurrence of cancer in the bones, up to 30 years may elapse. Throughout this timeframe, a typical patient undergoes many treatments that have effects on the bone microenvironment. Therefore, this review explores the clinical course of a representative patient with hormone receptor-positive bone metastatic breast cancer, examining key treatment options at each stage and their effects on preventing and treating bone metastases.

Extracellular Vesicles and Bone-Associated Cancer.

PURPOSE OF REVIEW: In this review, we describe the biology of extracellular vesicles (EV) and how they contribute to bone-associated cancers. RECENT FINDINGS: Crosstalk between tumor and bone has been demonstrated to promote tumor and metastatic progression. In addition to direct cell-to-cell contact and soluble factors, such as cytokines, EVs mediate crosstalk between tumor and bone. EVs are composed of a heterogenous group of membrane-delineated vesicles of varying size range, mechanisms of formation, and content. These include apoptotic bodies, microvesicles, large oncosomes, and exosomes. EVs derived from primary tumors have been shown to alter bone remodeling and create formation of a pre-metastatic niche that favors development of bone metastasis. Similarly, EVs from marrow stromal cells have been shown to promote tumor progression. Additionally, EVs can act as therapeutic delivery vehicles due to their low immunogenicity and targeting specificity. EVs play critical roles in intercellular communication. Multiple classes of EVs exist based on size on mechanism of formation. In addition to a role in pathophysiology, EVs can be exploited as therapeutic delivery vehicles.

Preferential uptake of antibody targeted calcium phosphosilicate nanoparticles by metastatic triple negative breast cancer cells in co-cultures of human metastatic breast cancer cells plus bone osteoblasts.

Calcium phosphosilicate nanoparticles (CPSNPs) are bioresorbable nanoparticles that can be bioconjugated with targeting molecules and encapsulate active agents and deliver them to tumor cells without causing damage to adjacent healthy tissue. Data obtained in this study demonstrated that an anti-CD71 antibody on CPSNPs targets these nanoparticles and enhances their internalization by triple negative breast cancer cells in-vitro. Caspase 3,7 activation, DNA damage, and fluorescent microscopy confirmed the apoptotic breast cancer response caused by targeted anti-CD71-CPSNPs encapsulated with gemcitabine monophosphate, the active metabolite of the chemotherapeutic gemcitabine used to treat cancers including breast and ovarian. Targeted anti-CD71-CPSNPs encapsulated with the fluorophore, Rhodamine WT, were preferentially internalized by breast cancer cells in co-cultures with osteoblasts. While osteoblasts partially internalized anti-CD71-GemMP-CPSNPs, their cell growth was not affected. These results suggest that CPSNPs may be used as imaging tools and selective drug delivery systems for breast cancer that has metastasized to bone.

Novel Techniques to Study the Bone-Tumor Microenvironment.

Many cancers commonly metastasize to bone. After entering the bone, cancer cells can interact with surrounding stromal cells, which ultimately influences metastasis progression. Extracellular vesicles, direct cell contact and gap junctions, and cytokines are all mechanisms of intercellular communication that have been observed to occur in the bone microenvironment. These methods of cellular crosstalk can occur between cancer cells and a variety of stromal cells, with each interaction having a different impact on cancer progression. Communication between cancer cells and bone-resident cells has previously been implicated in processes such as cancer cell trafficking and arrest in bone, cancer cell dormancy, cancer cell reactivation, and proliferation. In this chapter we review innovative techniques and model systems that can be used to study bidirectional crosstalk between cancer cells and stromal cells in the bone, with an emphasis specifically on bone-metastatic breast cancer. Investigating how metastatic cancer cells interact with, and are influenced by, the bone microenvironment is crucial to better understanding of the progression of bone metastasis.

Osteoblasts are "educated" by crosstalk with metastatic breast cancer cells in the bone tumor microenvironment.

INTRODUCTION: In a cancer-free environment in the adult, the skeleton continuously undergoes remodeling. Bone-resorbing osteoclasts excavate erosion cavities, and bone-depositing osteoblasts synthesize osteoid matrix that forms new bone, with no net bone gain or loss. When metastatic breast cancer cells invade the bone, this balance is disrupted. Patients with bone metastatic breast cancer frequently suffer from osteolytic bone lesions that elicit severe bone pain and fractures. Bisphosphonate treatments are not curative. Under ideal circumstances, osteoblasts would synthesize new matrix to fill in erosion cavities caused by osteoclasts, but this is not what occurs. Our prior evidence demonstrated that osteoblasts are diverted from laying down bone matrix to producing cytokines that facilitate breast cancer cell maintenance in late-stage disease. Here, we have new evidence to suggest that there are subpopulations of osteoblasts in the tumor niche as evidenced by their protein marker expression that have distinct roles in tumor progression in the bone. METHODS: Tumor-bearing tibia of mice was interrogated by immunofluorescent staining for the presence of osteoblasts and alterations in niche protein expression. De-identified tissue from patients with bone metastatic breast cancer was analyzed for osteoblast subpopulations via multi-plex immunofluorescent staining. Effects of breast cancer cells on osteoblasts were recapitulated in vitro by osteoblast exposure to breast cancer-conditioned medium. Triple-negative and estrogen receptor-positive breast cancer proliferation, cell cycle, and p21 expression were assessed upon contact with "educated" osteoblasts. RESULTS: A subpopulation of osteoblasts was identified in the bone tumor microenvironment in vivo of both humans and mice with bone metastatic breast cancer that express RUNX2/OCN/OPN but is negative for IL-6 and alpha-smooth muscle actin. These tumor "educated" osteoblasts (EOs) have altered properties compared to "uneducated" osteoblasts and suppress both triple-negative and estrogen receptor-positive breast cancer cell proliferation and increase cancer cell p21 expression. EO effects on breast cancer proliferation were mediated by NOV and decorin. Importantly, the presence of EO cells in the tibia of mice bearing tumors led to increased amounts of alkaline phosphatase and suppressed the expression of inflammatory cytokines in vivo. CONCLUSIONS: Our work reveals that there is a subpopulation of osteoblasts in the bone tumor microenvironment that demonstrate a functional role in retarding breast cancer cell growth.

Tumor-associated stromal cells as key contributors to the tumor microenvironment.

The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells.

'Educated' Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment.

Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-ß ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.

Printing the Pathway Forward in Bone Metastatic Cancer Research: Applications of 3D Engineered Models and Bioprinted Scaffolds to Recapitulate the Bone-Tumor Niche.

Breast cancer commonly metastasizes to bone, resulting in osteolytic lesions and poor patient quality of life. The bone extracellular matrix (ECM) plays a critical role in cancer cell metastasis by means of the physical and biochemical cues it provides to support cellular crosstalk. Current two-dimensional in-vitro models lack the spatial and biochemical complexities of the native ECM and do not fully recapitulate crosstalk that occurs between the tumor and endogenous stromal cells. Engineered models such as bone-on-a-chip, extramedullary bone, and bioreactors are presently used to model cellular crosstalk and bone-tumor cell interactions, but fall short of providing a bone-biomimetic microenvironment. Three-dimensional bioprinting allows for the deposition of biocompatible materials and living cells in complex architectures, as well as provides a means to better replicate biological tissue niches in-vitro. In cancer research specifically, 3D constructs have been instrumental in seminal work modeling cancer cell dissemination to bone and bone-tumor cell crosstalk in the skeleton. Furthermore, the use of biocompatible materials, such as hydroxyapatite, allows for printing of bone-like microenvironments with the ability to be implanted and studied in in-vivo animal models. Moreover, the use of bioprinted models could drive the development of novel cancer therapies and drug delivery vehicles.

Stromal-Derived Extracellular Vesicles Suppress Proliferation of Bone Metastatic Cancer Cells Mediated by ERK2.

Bone is a common site of cancer metastasis, including cancers such as breast, prostate, and multiple myeloma. Disseminated tumor cells (DTC) shed from a primary tumor may travel to bone and can survive undetected for years before proliferating to form overt metastatic lesions. This period of time can be defined as metastatic latency. Once in the metastatic microenvironment, DTCs engage in intercellular communication with surrounding stromal cells, which can influence cancer cell survival, proliferation, and ultimately disease progression. The role of the surrounding tumor microenvironment in regulating DTC fate is becoming increasingly recognized. We have previously shown that in the bone microenvironment, osteoblasts are "educated" by interactions with breast cancer cells, and these "educated" osteoblasts (EO) produce soluble factors that regulate cancer cell proliferation. In this study, we provide evidence indicating that EOs produce small extracellular vesicles (sEV) that suppress breast cancer proliferation, in part through regulation of ERK1/2 signaling. In addition, using EdU-incorporation assays and propidium iodide staining we demonstrate that exposure to EO-derived sEVs decreases breast cancer cell entry to S-phase of cell cycle. We also have evidence that particular microRNAs, including miR-148a-3p, are enriched in EO-derived sEVs, and that miR-148a-3p is capable of regulating breast cancer proliferation. IMPLICATIONS: These findings underscore the importance of sEV-mediated communication in the earlier stages of cancer progression, and suggest that EO-derived sEVs may be one mechanism by which the bone microenvironment suppresses breast cancer cell proliferation.

Combined Targeting of PARG and Wee1 Causes Decreased Cell Survival and DNA Damage in an S-Phase-Dependent Manner.

The DNA damage response (DDR) pathway sets the stage for tumorigenesis and provides both an opportunity for drug efficacy and resistance. Therapeutic approaches to target the DDR pathway include aiming to increase the efficacy of cytotoxic chemotherapies and synergistic drug strategies to enhance DNA damage, and hence cell death. Here, we report the first preclinical evaluation of a novel synergistic approach by using both genetic and small-molecule inhibition methods of silencing the DDR-related protein, poly (ADP-ribose) glycohydrolase (PARG), and the checkpoint kinase inhibitor, Wee1, in pancreatic ductal adenocarcinoma (PDAC) and colorectal carcinoma cells in vitro and in vivo. Mechanistically, we demonstrate that coinhibition of PARG and Wee1 synergistically decreased cell survival and increased DNA damage in an S-phase-dependent manner. IMPLICATIONS: In preclinical models, we demonstrate the efficacy and mechanism of action of targeting both PARG and Wee1 in PDAC and colorectal carcinoma cells. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/2/207/F1.large.jpg.

Osteoblasts are a major source of inflammatory cytokines in the tumor microenvironment of bone metastatic breast cancer.

Metastatic breast cancer cells co-opt the cells of the bone to increase their production of inflammatory cytokines. Here, we sought to identify key cytokines expressed by osteoblasts in vitro and in vivo in the presence of MDA-MB-231 metastatic breast cancer cells, including a bone-seeking variant. We hypothesized that osteoblast-derived cytokines increase in the presence of metastatic breast cancer cell conditioned medium (CM), act as chemoattractants for cancer cells, and enhance osteoclast formation. We detected increases in the concentrations of osteoblast-derived IL-6, MCP-1, VEGF, MIP-2, and KC in vitro in culture supernatants from MC3T3-E1 cells in the presence of metastatic breast cancer cell CM and from cancer-bearing femurs ex vivo. A comparison of cancer cell- and osteoblast-derived cytokines revealed that while breast cancer cells expressed the same or equivalent cytokines as the osteoblasts, the breast cancer cells only produced picogram quantities of MCP-1; osteoblasts expressed nanogram amounts. Bone-derived MCP-1 increased in the proximal metaphysis, an area where breast cancer cells preferentially trafficked following intracardiac inoculation in athymic mice. An MDA-MB-231 bone-seeking variant was not different from parental lines. Osteoblast CM was a potent chemoattractant for metastatic breast cancer cells. Furthermore, culture supernatants of osteoblasts treated with breast cancer cell CM enhanced osteoclast formation. These findings suggest that bone metastatic breast cancer cells utilize osteoblast-derived cytokines to facilitate breast cancer cell colonization and survival upon arrival in the bone microenvironment.

Immortalized, pre-malignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA.

INTRODUCTION: During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents', simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy. METHODS: The glands of 3-week old immune competent Balb/C female mice were utilized intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells which retain their template DNA. Nulliparous mice were then either injected with [(3)H]-thymidine ((3)H-TdR) to distinguish 5-BrdU-label retaining cells that enter the cell cycle and euthanized, or mated, injected with (3)H-TdR, and euthanized at various days post-coitus. Sections were stained for estrogen receptor-α(ER-α) or progesterone receptor (PR) via immunohistochemistry. Cells labelled with both 5-BrdU and (3)H-TdR were indicative of label-retaining epithelial cells (LREC). RESULTS: Cells that retained a 5-BrdU label and cells labelled with [(3)H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labelled nuclei (LREC) were found in the intact mammary gland of both pregnant and nulliparous mice, and in mammary glands implanted with pre-malignant cells. Double-labelled cells ((3)H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label ((3)H-TdR) to daughter cells; and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary implant groups. CONCLUSIONS: The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.

Ex-vivo analysis of the bone microenvironment in bone metastatic breast cancer.

In humans, breast cancer has a predilection to metastasize to the skeleton. While the mechanism for preferential metastasis is unknown, the bone microenvironment likely provides a fertile soil for metastatic breast cancer cells. In order to examine the bone microenvironment ex-vivo following the formation of breast cancer metastases, several techniques may be employed: fluorescence stereomicroscopy, magnetic resonance imaging (MRI), microCT (microCT), immunohistochemistry, and cytokine arrays, to name a few. These methods allow for a comprehensive evaluation of the bone microenvironment during bone metastatic breast cancer. By identifying alterations in the bone niche caused by metastatic breast cancer cells, it may be possible to block or disrupt these factors through the use of targeted drugs. Appropriate therapeutic treatment would allow for an improved quality of life and longer survival time for individuals with bone metastatic breast cancer.

The Bone Extracellular Matrix as an Ideal Milieu for Cancer Cell Metastases.

Bone is a preferential site for cancer metastases, including multiple myeloma, prostate, and breast cancers.The composition of bone, especially the extracellular matrix (ECM), make it an attractive site for cancer cell colonization and survival. The bone ECM is composed of living cells embedded within a matrix composed of both organic and inorganic components. Among the organic components, type I collagen provides the tensile strength of bone. Inorganic components, including hydroxyapatite crystals, are an integral component of bone and provide bone with its rigidity. Under normal circumstances, two of the main cell types in bone, the osteoblasts and osteoclasts, help to maintain bone homeostasis and remodeling through cellular communication and response to biophysical signals from the ECM. However, under pathological conditions, including osteoporosis and cancer, bone remodeling is dysregulated. Once in the bone matrix, disseminated tumor cells utilize normal products of bone remodeling, such as collagen type I, to fuel cancer cell proliferation and lesion outgrowth. Models to study the complex interactions between the bone matrix and metastatic cancer cells are limited. Advances in understanding the interactions between the bone ECM and bone metastatic cancer cells are necessary in order to both regulate and prevent metastatic cancer cell growth in bone.

Cancer Metastases to Bone: Concepts, Mechanisms, and Interactions with Bone Osteoblasts.

The skeleton is a unique structure capable of providing support for the body. Bone resorption and deposition are controlled in a tightly regulated balance between osteoblasts and osteoclasts with no net bone gain or loss. However, under conditions of disease, the balance between bone resorption and deposition is upset. Osteoblasts play an important role in bone homeostasis by depositing new bone osteoid into resorption pits. It is becoming increasingly evident that osteoblasts additionally play key roles in cancer cell dissemination to bone and subsequent metastasis. Our laboratory has evidence that when osteoblasts come into contact with disseminated breast cancer cells, the osteoblasts produce factors that initially reduce breast cancer cell proliferation, yet promote cancer cell survival in bone. Other laboratories have demonstrated that osteoblasts both directly and indirectly contribute to dormant cancer cell reactivation in bone. Moreover, we have demonstrated that osteoblasts undergo an inflammatory stress response in late stages of breast cancer, and produce inflammatory cytokines that are maintenance and survival factors for breast cancer cells and osteoclasts. Advances in understanding interactions between osteoblasts, osteoclasts, and bone metastatic cancer cells will aid in controlling and ultimately preventing cancer cell metastasis to bone.

Kinetics of metastatic breast cancer cell trafficking in bone.

PURPOSE: In vivo studies have focused on the latter stages of the bone metastatic process (osteolysis), whereas little is known about earlier events, e.g., arrival, localization, and initial colonization. Defining these initial steps may potentially identify the critical points susceptible to therapeutic intervention. EXPERIMENTAL DESIGN: MDA-MB-435 human breast cancer cells engineered with green fluorescent protein were injected into the cardiac left ventricle of athymic mice. Femurs were analyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry, and histomorphometry at times ranging from 1 hour to 6 weeks. RESULTS: Single cells were found in distal metaphyses at 1 hour postinjection and remained as single cells up to 72 hours. Diaphyseal arrest occurred rarely and few cells remained there after 24 hours. At 1 week, numerous foci (2-10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 weeks, fewer but larger foci (> or =50 cells) were seen. Most bones had a single large mass at 4 weeks (originating from a colony or coalescing foci) which extended into the diaphysis by 4 to 6 weeks. Little change (<20%) in osteoblast or osteoclast numbers was observed at 2 weeks, but at 4 to 6 weeks, osteoblasts were dramatically reduced (8% of control), whereas osteoclasts were reduced modestly (to approximately 60% of control). CONCLUSIONS: Early arrest in metaphysis and minimal retention in diaphysis highlight the importance of the local milieu in determining metastatic potential. These results extend the Seed and Soil hypothesis by demonstrating both intertissue and intratissue differences governing metastatic location. Ours is the first in vivo evidence that tumor cells influence not only osteoclasts, as widely believed, but also eliminate functional osteoblasts, thereby restructuring the bone microenvironment to favor osteolysis. The data may also explain why patients receiving bisphosphonates fail to heal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblast function are needed to effectively treat osteolytic bone metastases.

Understanding Mitochondrial Polymorphisms in Cancer.

Alterations in mitochondrial DNA (mtDNA) were once thought to be predominantly innocuous to cell growth. Recent evidence suggests that mtDNA undergo naturally occurring alterations, including mutations and polymorphisms, which profoundly affect the cells in which they appear and contribute to a variety of diseases, including cardiovascular disease, diabetes, and cancer. Furthermore, interplay between mtDNA and nuclear DNA has been found in cancer cells, necessitating consideration of these complex interactions for future studies of cancer mutations and polymorphisms. In this issue of Cancer Research, Vivian and colleagues utilize a unique mouse model, called Mitochondrial Nuclear eXchange mice, that contain the nuclear DNA from one inbred mouse strain, and the mtDNA from a different inbred mouse strain to examine the genome-wide nuclear DNA methylation and gene expression patterns of brain tissue. Results demonstrated there were alterations in nuclear DNA expression and DNA methylation driven by mtDNA. These alterations may impact disease pathogenesis. In light of these results, in this review, we highlight alterations in mtDNA, with a specific focus on polymorphisms associated with cancer susceptibility and/or prognosis, mtDNA as cancer biomarkers, and considerations for investigating the role of mtDNA in cancer progression for future studies. Cancer Res; 77(22); 6051-9. ©2017 AACR.

Mathematical modeling of material-induced blood plasma coagulation.

Contact activation of the intrinsic pathway of the blood coagulation cascade is initiated when a procoagulant material interacts with coagulation factor XII, (FXII) yielding a proteolytic enzyme FXIIa. Procoagulant surface properties are thought to play an important role in activation. To study the mechanism of interaction between procoagulant materials and blood plasma, a mathematical model that is similar in form and in derivation to Michaelis-Menten enzyme kinetics was developed in order to yield tractable relationships between dose (surface area and energy) and response (coagulation time (CT)). The application of this model to experimental data suggests that CT is dependent on the FXIIa concentration and that the amount of FXIIa generated can be analyzed using a model that is linearly dependent on contact time. It is concluded from these experiments and modeling analysis that the primary mechanism for activation of coagulation involves autoactivation of FXII by the procoagulant surface or kallikrein-mediated reciprocal activation of FXII. FXIIa-induced self-amplification of FXII is insignificant.

Procoagulant stimulus processing by the intrinsic pathway of blood plasma coagulation.

Potentiation of the intrinsic pathway of human blood plasma coagulation in vitro by contact with a solid procoagulant surface leads to bolus release of thrombin (FIIa) in concentration proportion to the intensity of activation as measured by procoagulant surface area or energy (water wettability). This rather remarkable finding is confirmed using two different assays: one triggering coagulation substantially through the intrinsic pathway alone and the second triggering coagulation through the intrinsic pathway in the presence of exogenous FIIa spikes. Similarity of experimental outcomes of these assays strongly suggests that endogenous FIIa production through the intrinsic pathway is independent of the absolute amount of FIIa present in plasma. Furthermore, we corroborate previous work indicating that procoagulant surfaces remain activating after repeated use and are not poisoned or denatured in the process of activating plasma coagulation. It is concluded that the sharp control mechanism that gives rise to bolus-production of FIIa from the intrinsic pathway must occur between surface activation of FXII and the FII --> FIIa step, is not related to inhibition by FIIa, and does not involve deactivation of procoagulant surfaces.